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Immune Priming with Ultrasound
Conflicts and Grants
- NIH (R01 EB009040)
- NCI SBIR grant with
Celldex Therapeutics, Inc.
- Project Energy with
Johnson & Johnson
Chandan Guha, MBBS, PhD
Professor and Vice Chair, Radiation Oncology
Professor, Urology and Pathology
Director, Einstein Institute of Onco-Physics
Montefiore Medical Center
Albert Einstein College of Medicine, Bronx, NY
Mutated Cells “Killing” tumor
Tumor Ablation
Tumor
progression
Diagnosis
Primary => Mets
Immune
Surveillance
Living “with”
Mutated Cells
Immune
Escape (Epigenetic loss – Antigen presentation)
Immune
Restoration
Resurgence
Living “with”
Tumor
Tumor Evolution
• Cancer cure restored
immune surveillance
• Ablation with Immune
Restoration is curative
Suppression (IL10, TGFß, PD-L1)
Cooption (Macrophage, Ectopic Lymphoid structure)
“Focal Therapy for Systemic Cure”
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)
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Project ENERGY.01: Proposed Study Design
Immuno-PrimingAbla on(IPA)Therapies
Immuno-Priming (IP)
LOFU1-2 HT RP IRE1-2 RFP1-2 Digoxin Metformin
Immuno-Priming - Ablation (IPA)
SBRT (10-20 Gy) RFA
Energy IPA1-2 +
GM-CSF +/-
Anti-PD1
Metastatic Model
3LL Lewis Lung
GEM models
RapidCap
Liver (Ch)
KPC
LOFU / IRE / Digoxin + +
SBRT (10-20 Gy) RFA
2 Metastatic (3LL, 4T1)
1 GEM (CaP / KPC)
LOFU Low Energy / Intensity Focused Ultrasound
HT Histotripsy
RP Radiation Priming
IRE Irreversible Electroportaion
RFP Radiofrequency Priming
SBRT Stereotactic Body Radiation
Therapy
RFA Radio Frequency Ablation
Study 1: 3LLIP only; Biomarker endpoints
Study 2: 3LLIPA; Biomarker, Immune Profile,
Tumor control and survival endpoints
Study 3: 3LL; 4T1;GEMIPA + DC Act./Check-Point Inhib.;
Biomarker, Tumor control and survival endpoints
Best 2 IPA’s
Best 2 of all
Go/No-Go for IRE/RFAGo/No-Go for LoFu Licensing
Internal R&D (Ethicon)External R&D (Electroblate, Varian)
NewCo Formation
=
Thorsten Melcher
Autologous in situ tumor vaccines
3 weeks
3 weeks
3 weeks
Flt3L (10 µg/day)
X 10 days
• RT (60 Gy)
• Surgery
No Treatment
Flt3L (10 µg/day)
X 10 days
• RT (60 Gy)
• Surgery
Treatment Protocol
Survival Time (Days)
20018016014012010080604020
1
.8
.6
.4
.2
0
Flt3LRT + Flt3LRTControl
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RT + Flt3L Improves Survival of Tumor-bearing C57Bl/6 Mice
Survival Time (Days)
20018016014012010080604020
1
.8
.6
.4
.2
0
Flt3LRT + Flt3LRTControl
RT + Flt3L
Survival Time (Days)
C57Bl/6 mice
(RT+Flt3L - 55% cured)
ImmunodeficientNude mice
(RT+Flt3L - 0% cured)
Survival Time (Days)
Kaplan & Meier Survivorship Function
16012080400
1
.8
.6
.4
.2
0
D2 - RT + Flt3L-30
D1 - RT + Flt3L-10
E - S + Flt3L
C - Surgery
B - RT
Systemic Effects of Primary Tumor Irradiation
Surgery + Flt3L
Sample size: 29 patients
8/1/2017
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Sample size: 29 patients
Nitin Ohri
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
173 year-old
Korean male
Right lung squamous
cell carcinoma with
multiple lung masses
and bilateral
mediastinal adenopathy
Carboplatin + gemcitabine
50 Gy in 5
fractions to
RLL mass
Progression at 6
weeks, death at 4
months
255 year-old
Hispanic female
Right lung
adenocarcinoma, with
bilateral lung nodules
Chemoradiotherapy for
localized disease,
nivolumab for metastatic
disease
34 Gy in 1
fraction to
LUL nodule
Partial response
at 2 months,
stable at 4 months
3
80 year-old
Caucasian
female
Right lung squamous
cell carcinoma with
spine and pelvic
metastases
Chemoradiotherapy for
localized disease,
carboplatin + gemcitabine
for metastatic disease,
nivolumab
50 Gy in 5
fractions to
right lung
mass
Partial response
at 2 months
4
73 year-old
Caucasian
female
Right lung
adenocarcinoma with
mediastinal adenopathy
and liver metastasis
Carboplatin/ +
pemetrexed, maintenance
pemetrexed
50 Gy in 5
fractions to
RLL mass
PET/CT on 4/27
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/CTTarget 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
Immunotherapy
Tumor Models End Points Limitation
Single
Fraction
SFRT
25-60 Gy
Flt3L
+ / -
CD40L
• Lewis Lung
3LL in C57/Bl6
• BN1LNE Liver
(HCC) in
Balb/c
1. Primary Tumor
Growth
2. Metastases
3. Survival
4. Immune assays
• Murine Ectopic
Transplantation
Models
• RT Dose
• Fractionation
• RT to Draining
Lymph Node
• Break Tolerance
to self antigens
• Small Animal
Treatment
• Lack of Immune
Surveillance and
carcinogenic
environment
20 Gy TLR9
agonist
• Lewis Lung
3LL in C57/Bl6
PTG, Mets, Survival
10 Gy Listeria-
PSA
ADXS31-
142
• TRAMPC1
TPSA23
Prostate
Cancer
PTG and Immune
Assays
20Gy x 3 PD1-Fc • Lewis Lung
3LL in C57/Bl6
PTG, Mets, Survival
LOFU HIFU • TPSA23
Prostate
PTG, Immune
Assays
LOFU SBRT
(10 Gy x 3)
• B16 Melanoma PTG, Mets, Survival
Immune Assays
8/1/2017
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Acoustic priming
1 W/cm2
2000 W/cm2
HIFU Beam
TM
ULTRASOUND -- ADVANTAGES
• US Can do both Imaging
and Treatment
• Quick Tissue Destruction
• Bloodless
• Precise and Accurate
• Non-sterile environment
Diagnostic (Imaging) Beam
Focal Zone
Peripheral Zone
HIFU directed harmlessly across skin and rectum toward the tumor
8/1/2017
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• 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
Therapeutic Ultrasound as an autologous in situ
tumor vaccine
Condition #1
Condition #2 Condition #3 Condition #4 Condition #5
Duty Cycle (%) 1 25 50 75 100
Power (W) 32 16 8 4 2
Time (ms) 1000 625 1250 2500 5000
Thermal Energy (J)
0.32 2.5 5 7.5 10
Peak NegativePressure
(MPa)8.14 6.08 4.58 3.34 2.46
Mechanical
Energy
Thermal
Energy
LOFU parameters
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
The “sonic stress” of LOFU
8/1/2017
8
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
0.000
1.000
2.000
3.000
4.000
5.000
6.000
7.000
8.000
9.000
10.000
CSF
2RB
DN
AJB
1
HSP
H1
HSP
E1
HSP
B1
HSP
D1
HSP
A4L
HSP
A6
HSP
A7
HSP
90A
A1
HSP
90A
A4P
DN
AJA
4
AN
KR
D1
IL8
JUN
ATF
3
Control
Treated
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.
-20.00
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
mR
NA
Fo
ld C
han
ge
Hspa1a
132.37
Hspa1b
80.77
Hsp expression
0
10
20
30
40
50
60
70
80
0 5 10 15 20 25 30
Time post-LOFU (h)
mR
NA
fo
ld in
cre
ase
Hspa1b
Hspb1-1
Hspb1-2
Hspd1
Hsph1
Hsp90aa1
Dnajb5
Increase in Hsp70 mRNA expression after LOFU treatment
8/1/2017
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Immunomodulation of tumor cells
Surface Calreticulin (CRT) Intracellular HSP70
In vitro Effect of LOFU on Cell Surface Markers
3 Watt LoFu 5 Watt LoFu
3LL 4T1 TPSA23 3LL 4T1 TPSA23
6h 24h 6h 24h 6h 24h 6h 24h 6h 24h 6h 24h
ER Stress
HSP70
CRT
BiP
Co-StimulatoryMHC I
CD86
Death Receptors
Fas
CD40
Inhibitory PD-L1
0 5 10 20 30No change TBD
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
SRS
Ch-RT
“eat me”signal
“danger”signal
Induce Cell death
Calreticulin membrane
Translocation
HSP activation
HMGB1 release
TL4 binding
LOFU
8/1/2017
10
– Reprogram tolerogenic DCs (IDO inhibitors)
– Inhibit regulatory T cells (Treg)
– Reverse T-cell anergy in TDLN – LOFU primary tumor
Treat the tumor draining lymph node (TDLN – immune privilege site)
T cell activation vs. T cell anergy
CD4+ T cell
Full stimulation
Cytokine productionCell Proliferation
Re-stimulation
Cytokine productionCell Proliferation
T cell Activation
Re-stimulation
Hyporesponsive phenotype
Hyporesponsive phenotype
Anergic stimulus
CD4+ T cell
T cell Anergy
CD3 CD28
T cell Activation vs. T cell Anergy
Anergic StimulusFull Activation
Ca
NFATp
p pp pp
p
p
MAP kinases CaCa
NF-kBAP-1
NFATp50 p65
Activation-induced genes
Ca
NFATp
p pp pp
p
p
CaCa
Co-partners
Anergy-inducing genes
?
TCR CD28 TCR
NFAT
8/1/2017
11
0
5
10
15
20
No Tumur T-DLN T-NDLN
IL-2
(n
g/m
L)
No Tumor
***
**
*
0
2
4
6
8
10
No Tumor T-DLN T-NDLN
IFNg
(ng
/mL
)
*****
**
0
5
10
15
20
25
30
No Tumor T-DLN T-NDLN
IL-2
(n
g/m
L)
0
2
4
6
8
10
12
No Tumor T-DLN T-NDLN
IFNg
(ng
/mL
)
0
2
4
6
8
No Tumour T-DLN T-NDLN
IFNg
(ng
/mL
)
No-Tumor
***
**
0
3
6
9
12
15
18
No Tumour T-DLN T-NDLN
IL-2
(n
g/m
L)
No-Tumor
B16 in B6 mice B16-OVA in OT-II mice B16 in Tyrp1 mice
B16 tumors induced CD4+ T cell anergy
0
10
20
30
40
IL-2
(n
g/m
L)
NDLN DLN
*
LOFU prevents B16-induced CD4+ T cell anergy
T cell anergy: transcriptional program
8/1/2017
12
0
4
8
12
Untreated LOFU
Fo
ld In
du
cti
on
mR
NA
Grail
0
2
4
6
Untreated LOFU
Itch
0
1
2
3
4
Untreated LOFU
Cbl-b
0
2
4
6
Untreated LOFU
Fo
ld In
du
cti
on
mR
NA
Grg4
0
1
2
3
4
5
Untreated LOFU
Egr2
LOFU prevents B16-induced CD4+ T cell anergy
0
50
100
150
200
250
Resting Stimulated
IL-2
(n
g/m
L)
Control Th1
Anergic Th1
0
1
2
3
4
5
IL-2
(n
g/m
L)
DC
Anergic T cells
Untr. lysate
LOFU lysate
**
+ + + +++ +−
+− −−+− −−
Tyrp1 CD4 Th1 cellsAnergized in vitro
LOFU can reverse established T cell anergy
LOFU as immune adjuvant for IGRT
B16-M1Melanoma
LOFU
IGRT
0 1 0
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
2 0 3 0 4 0 5 0 6 0
T im e (d a y s )
Vo
lum
e (
mm
3)
0 1 0
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
2 0 3 0 4 0 5 0 6 0
T im e (d a y s )
Vo
lme
(m
m3
)
U nt
LO FU
0 1 0
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
2 0 3 0 4 0 5 0 6 0
T Im e (d a y s )
Vo
lum
e (
mm
3)
U nt
LO FU
RT
0 1 0
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
2 0 3 0 4 0 5 0 6 0
T im e (d a y s )
Vo
lum
e (
mm
3)
U nt
LO FU
L O F U + R T
RT
8/1/2017
13
LOFU as adjuvant for IGRT requires T cells
0 2 0 4 0 6 0
0
2 0 0
4 0 0
6 0 0
8 0 0
L O F U
L O F U + R T
U n t
R T
T im e (d a y s )
Vo
lum
e (
mm
3)
Nude miceB16 melanoma
0 1 0
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
2 0 3 0 4 0 5 0 6 0
T im e (d a y s )
Vo
lum
e (
mm
3)
U nt
LO FU
L O F U + R T
RT
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
[LOFU (3W) + RT (10 Gy)] X 3
A marked shift from naïve to activated phenotype was observed in the combination treatmentTILs.
8/1/2017
14
CD62L-VE/CD8+VE in Tumor infiltrating lymphocytes (TILs)
No Treatment
[LOFU (3W) + RT (10 Gy)] X 3
LOFU+RT treatment causes a significant increase in activated CD8 T-cells in TILs
Oncoscience434www.impactjournals.com/oncoscience
www.impactjournals.com/oncoscience/ Oncoscience 2014, Vol.1, No.6
Low intensity focused ultrasound (LOFU) modulates unfolded protein response and sensitizes prostate cancer to 17AAG
Subhrajit Saha1, Payel Bhanja1, Ari Partanen2, Wei Zhang1, Laibin Liu1, Wolfgang A. Tomé1,4 and Chandan Guha1,3,4
1 Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York, USA
2 Philips Healthcare, Cleveland, OH, USA
3 Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, USA
4 Montefio
r
e Me di cal Cent er , New York, NY, USA
Correspondence to: Chandan Guha, email: [email protected]
Keywords: Prostate Cancer, 17AAG, Focused Ultrasound, UPR, ER stress
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
The hypoxic tumor microenvironment generates oxidative Endoplasmic Reticulum
(ER) stress, resulting in protein misfolding and unfolded protein response (UPR).
UPR induces several molecular chaperones including heat-shock protein 90 (HSP90),
which corrects protein misfolding and improves survival of cancer cells and resistance
to tumoricidal therapy although prolonged activation of UPR induces cell death. The
HSP90 inhibitor, 17AAG, has shown promise against various solid tumors, including
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,
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
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
response in tumors resulting in significa nt induction of apoptosis and tumor growth
retardation. These observations suggest that LOFU-induced ER stress makes the
ultrasound-treated tumors more susceptible to chemotherapeutic agents, such as
17AAG. Thus, a novel therapy of LOFU-induced chemosensitization may be designed
for locally advanced and recurrent tumors.
INTRODUCTION
Therapeutic ultrasound is being developed as an
image-guided ablative treatment for solid tumors. High-
intensity focused ultrasound (HIFU) delivers sonic energy
to a small, well-defin
e
d target region in malignant tissue,
causing a rapid rise in tissue temperature exceeding
80-90oC, thereby inducing instantaneous coagulative
necrosis at the focal point. HIFU is quickly emerging as
an effective image-guided, minimally invasive treatment
modality for solid tumors, including prostate cancer (PC)
[1, 2]. The biological effects of therapeutic ultrasound
results from both thermal and non-thermal/mechanical
bioeffects, which arise from complex interactions of
propagating ultrasound waves with target tissue[3] .
Thermal effects are due to ultrasound absorption and
conversion to heat through vibrational excitation of
tissue, leading to localized temperature elevation.
Mechanical bioeffects that are unique to HIFU include
acoustic radiation forces and acoustic cavitation[4, 5].
HIFU may pose a risk to normal tissues, e.g., bones,
blood vessels, and nerves adjacent to target malignant
tissue, due to rapid temperature elevation leading to
nearly instantaneous thermal coagulation at high acoustic
intensities[5]. Furthermore, bubble activity mediated
acoustic cavitation may be induced at high acoustic
pressure levels, leading to locally induced stress and high
energy release, possibly resulting in and assisting thermal
Oncoscience439www.impactjournals.com/oncoscience
unchanged with LOFU or 17AAG or the combination
therapy (Figure 4A & C), indicating that macroautophagy
was not altered with ultrasound therapy. However, LOFU
alone or 17AAG alone induced the expression of LAMP-
2A (Figure 4A & B), indicating a compensatory increase in
CMA after therapies that increase the burden of misfolded
proteins in the ER. Interestingly, the combination of
LOFU+17AAG inhibited the levels of LAMP-2A below
the basal levels seen in these tumors. This suggests that the
Figure 6: LOFU+17AAG treatment reduces the
expression of prostate cancer stem cell markers in
RM1 cells. Flow cytometry of isolated RM1 tumor cells
showed signific
a
nt decrease in SCA1 (A & B), CD44 (A &
C), CD133 (A & D), and α2β1 integrin (A & E) cell surface
expression on RM1 tumor cells after LOFU+17AAG treatment.
(F) qRT-PCR array followed by heat map analysis showed that
LOFU+17AAG combination treatment group down-regulates
the mRNA levels of stem cell transcription factors.
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 five fractions administered over
two weeks. Animals received 17AAG three times a week during
this time. Tumors were harvested 24 hours after the last fraction
of LOFU. B. RM1 tumor. In C57Bl6 mice, LOFU+17AAG
combination treatment reduced RM1 tumor growth significa nt ly
(p<0.004), compared to controls. Note that either LOFU or
17AAG alone failed to control tumors significa nt ly . LOFU
sensitized the effects of a low dose (25mg/kg of body weight)
17AAG. C. PC3 tumor. In BalbC nu/nu mice LOFU+17AAG
combination treatment showed significa nt reduction in PC3
tumor growth (p<0.007).
Oncoscience439www.impactjournals.com/oncoscience
unchanged with LOFU or 17AAG or the combination
therapy (Figure 4A & C), indicating that macroautophagy
was not altered with ultrasound therapy. However, LOFU
alone or 17AAG alone induced the expression of LAMP-
2A (Figure 4A & B), indicating a compensatory increase in
CMA after therapies that increase the burden of misfolded
proteins in the ER. Interestingly, the combination of
LOFU+17AAG inhibited the levels of LAMP-2A below
the basal levels seen in these tumors. This suggests that the
Figure 6: LOFU+17AAG treatment reduces the
expression of prostate cancer stem cell markers in
RM1 cells. Flow cytometry of isolated RM1 tumor cells
showed signific
a
nt decrease in SCA1 (A & B), CD44 (A &
C), CD133 (A & D), and α2β1 integrin (A & E) cell surface
expression on RM1 tumor cells after LOFU+17AAG treatment.
(F) qRT-PCR array followed by heat map analysis showed that
LOFU+17AAG combination treatment group down-regulates
the mRNA levels of stem cell transcription factors.
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 five fractions administered over
two weeks. Animals received 17AAG three times a week during
this time. Tumors were harvested 24 hours after the last fraction
of LOFU. B. RM1 tumor. In C57Bl6 mice, LOFU+17AAG
combination treatment reduced RM1 tumor growth significa nt ly
(p<0.004), compared to controls. Note that either LOFU or
17AAG alone failed to control tumors significa nt ly . LOFU
sensitized the effects of a low dose (25mg/kg of body weight)
17AAG. C. PC3 tumor. In BalbC nu/nu mice LOFU+17AAG
combination treatment showed significa nt reduction in PC3
tumor growth (p<0.007).
Oncoscience438www.impactjournals.com/oncoscience
of the UPR. Indeed, TUNEL staining demonstrated that
LOFU induced minimal apoptosis over untreated controls.
Treatment with 17AAG induced significa nt apoptosis in
prostate tumors, which was further increased by LOFU
(p<0.004) (Figure 3E). Thus, 17AAG-mediated inhibition
of HSP90 and activation of CHOP by the combination
of LOFU+17AAG switched on apoptotic cell death of
prostate tumors.
LOFU+17AAG inhibits Chaperone Mediated
Autophagy (CMA) in tumor cells.
Degradation of misfolded proteins is mediated by
the proteosomal pathway and autophagy. Autophagy has
been implicated in the tumorigenesis process in a context-
dependent role, where it might provide amino acids and
other essential nutrients to the metabolic pathways of
hypoxic tumors that are nutrient deprived [22]. Indeed, an
increase in CMA activity has been described in a wide
variety of human tumors and CMA has been implicated
in survival, proliferation, and metastases of tumor cells
[23]. Therefore, we quantitated the levels of two key
proteins participating in autophagy, Beclin, a marker of
macroautophagy, and LAMP-2A lysosomal receptor, a
marker of CMA in the tumor tissues of various treatment
cohorts. As shown in Figure 5, Beclin levels remain
Figure 4: LOFU+17AAG treatment inhibits Chaperone
Mediated Autophagy (CMA) in RM1 tumor cells. (A &
B) Immunoblot analysis showed several fold down-regulation
of SMA marker LAMP2a expression level in combination
treatment group. Treatment with either LOFU or 17AAG up-
regulates the LAMP2a expression level. (A & C) Combination
treatment of LOFU and 17AAG did not alter the expression
level of Beclin, a macroautophagy marker.
Figure 3: LOFU+17AAG activates pro-apoptotic
pathways of UPR and induces apoptosis in tumor
cells. A & B. Western blot of pPERK (A) and peIF2a (B).
LOFU+17AAG activates PERK by phosphorylation of PERK
(pPERK), which further induces the phosphorylation of eIF2α
phosphorylation (peIF2α). C. Real Time-PCR analysis of CHOP
mRNA. There was a 25±1.3-fold increase in CHOP transcript
in LOFU+17AAG treated group, compared to control. D.
Real Time-PCR array of RNA isolated from LOFU+17AAG-
treated tumors. Heat map analysis showed that LOFU+17AAG
treatment group increased the transcript level of apoptotic genes
several folds compared to untreated control or LOFU groups.
E. TUNEL staining. Immunohistochemical staining showed
predominantly tunel positive cells in LOFU+17AAG treatment
group, compared to control or LOFU group. Note that 17AAG
alone also induced apoptosis in tumor tissue that was augmented
by LOFU.
100
101
102
103
104
0
20
40
60
80
100
SCA1
100
101
102
103
104
0
20
40
60
80
100
CD4410
010
110
210
310
40
20
40
60
80
100
CD133
LOFU+17AAG
LOFU+17AAG reduces the expression of prostate
cancer stem cell marker
Control
Did the number of cells go down or expression?
8/1/2017
15
Acoustic Priming
Immunotherapy
HIFU
RFA
Cryotherapy
Chemotherapy
Radiotherapy
Tumor
Sensitization of ablative
therapies
Chemosensitization
Radiosensitization
↑Immunosensitization
Autologous in situ tumor vaccines
The FOCAL Team
Medical Physics
Assoc.: Patrik Brodin
Director: Wolfgang Tome
Guha Laboratory
Students.:
Lorenzo Agoni
Karin Skalina
Talicia Savage
Justin Tang
Research Associates:
Huagang Zhang
Associates:
Indranil Basu
Lisa Scandiuzzi
Immune ToleranceSanmay Bandyopadhyay
Fernando Macian
Immune TherapeuticsSteve Almo
Clinical Trials
Rafi Kabariti
Nitin Ohri
Madhur Garg
Thank you!
8/1/2017
16
