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INTRATUMORAL ACETIC ACID INJECTION ERADICATES HUMAN
PROSTATE CANCER TUMORS IN A MURINE MODEL
Jasneet Singh Bhullar MD1, Gokulakkrishna Subhas MD1, Erina Kansakar MD1,
Jasper Gill BS2 , Boris Silberberg MD2, Jacqueline Tilak BS2, Lee Andrus LVT
BIS2, Milessa Decker LVT2, Patrick Hurley MD1, Jeffrey O’Connor MD1 and Vijay
K. Mittal MD FACS1
Departments of 1Surgery and 2Patient Care Research.
Providence Hospital and Medical Centers, Southfield, MI, USA
Address for correspondence
Jasneet Singh Bhullar MD
Department of Surgery
Providence Hospital & Medical Centers
16001 West Nine Mile Road,
Southfield, MI-48075, USA
Phone -248-436-8001
Fax – 248-849-5380
Email- [email protected]
Key words: Acetic acid, intratumoral injection, human prostate cancer cell lines,
prostate cancer, xenograft, murine model.
ABSTRACT
Purpose:
Prostate cancer treatment is associated with substantial morbidity. Ideal
treatment of localized prostate cancer would be an effective local therapy with
minimal morbidity. Direct injections have been used to treat benign prostatic
hyperplasia without major complications, but in limited cases. We evaluated the
local oncotoxic effects of acetic acid in a prostate cancer xenograft murine
model.
Methods:
PC3 and LNCaP human prostate cancer cell lines were used to grow
subcutaneous tumors in SCID mice. For each cell line, 14 mice underwent
intratumor injection with 25% acetic acid (0.05 ml/100 cm3 of tumor) after the
tumor was >300 mm3. Post treatment one mouse/group was euthanized after 2
hrs, 24 hrs, 1 week and 2 weeks; remaining mice (n=10) were sacrificed at 120
days. Control mice (8/group) were euthanized after they met the humane criteria
for tumor burden and overall health.
Results:
Tumor necrosis was noted immediately post injection; by 24 hrs, ulceration and
crusting of overlying skin were noted, which healed into scars by 23±5 days.
Histological examination showed tumor degeneration and necrosis with blood
vessel obstruction. Ten treated mice in both groups survived for 120 days, which
was much longer than the mean survival of PC3 (40±9 days) and LNCaP (56±10)
control mice.
Conclusions:
Direct injection of acetic acid successfully eradicated both tumors. This treatment
option could potentially be used in humans for treatment of early localized
prostate cancer and non-operative management of locally advanced cases. This
is the first report of successful local chemical therapy for prostate cancer.
Introduction:
In the United States alone, 200,000 men are diagnosed with prostate cancer
each year and one out of six men will be diagnosed in their lifetime (1). With
increasing awareness and regular cancer screening, the majority of newly
diagnosed prostatic cancers are localized and found at an early stage. In spite of
an earlier diagnosis, treatment of these cancers is associated with substantial
morbidity. More commonly, radical prostatectomy, brachytherapy, or external
beam radiotherapy are used to treat the early localized prostate cancer, but each
modality has its morbidity.
During early phases of the disease, the malignant prostatic lesions tend to
remain focal and restricted to the prostate gland itself. This, combined with the
anatomic accessibility of the prostate gland, makes direct intratumoral injection of
carcinotoxic and carcinostatic agents a real possibility for effective and relatively
noninvasive treatment (2).
Direct transurethral injection of dehydrated ethanol into the prostate under
endoscopic guidance and transperineal injection under transrectal ultrasound
guidance have been used to treat benign prostatic hyperplasia and prostatic
adenocarcinoma (3) in limited cases, resulting in improved micturition without
major complications.
Historically many chemicals - ethanol, carbolic acid, acetic acid and glycerin have been used alone or in combination to successfully chemo-ablate benign
prostatic hyperplasia by direct prostatic injections (4, 5, 6, 7). Ethanol injection
therapy for locally invasive prostatic cancer has been reported in a few cases (8)
as well as in a murine xenograft model, which showed regressed prostatic cancer
tumors (9). Currently, acetic acid is used to ablate hepatic carcinomas and other
malignancies (10, 11). This led to our hypothesis that intratumoral administration
of acetic acid could be an effective treatment for prostate cancer, and could be
safely used to inject the prostate in humans as it has been used to treat benign
hyperplasia.
In this study, based in part on promising in vitro results from our laboratory, we
explored the effectiveness of direct intratumoral injection of acetic acid in
malignant prostatic tumors. To our knowledge, this is the first examination of
intratumoral acetic acid as a treatment strategy for prostate cancer, and we feel
that these data form powerful preliminary evidence indicating that such a
minimally invasive strategy could be efficacious.
Material and Methods:
Cell lines:
PC3 and LNCaP human prostate cancer cells were obtained from ATCC
(Rockville, Maryland, USA). Cells were maintained at 37°C, 5% CO2 and 95%
humidity in DMEM medium (CellGro,Herndon, Virginia, USA) supplemented with
10% (v/v) heat inactivated fetal bovine serum (BioWhittaker, Walkersville,
Maryland, USA.
Mice:
Hairless male scid mice at 8 weeks of age were purchased from Charles River
Laboratories (Wilmington, Massachusetts, USA). Animals were allowed to
acclimate for 2 weeks prior to experimentation. The animals were cared and
managed in accordance with the National Institutes of Health Guide for the Care
and Use of Laboratory Animals.
Xenograft:
There were 22 mice in each of PC3 and LNCaP groups. In each cell line group 8
were control mice while 14 underwent treatment with acetic acid. PC3 and
LNCaP cells were grown to 70% confluence, harvested and injected in the
dorsum of animals subcutaneously. Each inoculum consisted of 100 μL of cell
suspension with 107 cells suspended in matrigel. Tumors were measured
biweekly. Tumor volume was determined using the following formula:
Volume = Length × Width2.
Direct intratumoral injection was done in 14 mice for each cell line group. Tumors
were injected with 25% acetic acid, 0.05ml/100cm3 of tumor after it attained a
size > 300mm3. Post treatment one mouse in each group was euthanized after 2
hours, 24 hours, 1 week and 2 weeks; the remaining mice (n=10) were allowed to
survive for 120 days. As a final measure of the potential usefulness of the
treatment as a component of prostate cancer management, we assayed the
ability of the intratumoral acetic acid injection protocol to extend the life of
animals in our prostate cancer xenograft model. Because they are growing
subcutaneously rather than orthotopically, xenograft tumors may grow to
significant size without causing animal death. For humane reasons, a scoring
system was established to assess animal welfare and animals not able to meet
any requirement were euthanized. The scoring system consisted of the following:
1. Maintenance of normal weight (Weight loss >15%)
2. Normal ambulation
3. Normal grooming
4. Normal feeding
Importantly, the decision to remove an animal from the protocol due to extreme
tumor burden was made by an animal care technician unaware of the treatment
group of the particular animal at the time of the decision. Thus, humane removal
of an animal from the protocol was recorded as a death event, and with these
data we evaluated survival.
After euthanizing, specimens from all animals were sent for histopathology and
the hematoxylin & eosin-stained slides were reviewed by a board certified
pathologist.
Results:
Control mice:
There were eight control mice each in the PC3 and LNCaP groups. The control
mice were monitored for tumor growth rate and overall health and were
euthanized according to the humane criteria described earlier, which was
recorded as a death event. The PC3 tumors grew faster and more solid tumors,
while the LNCaP tumors showed a slower rate of tumor growth and grew
spherical soft tumors (Picture -1).
The mean survival was 40±9 days and 56±10 in the PC3 and LNCaP group
control mice, respectively. Survival in the PC3 group was reduced because of the
faster tumor growth leading to greater tumor volume and its consequent effects
on the overall health of the mouse. No changes were noted on the skin over the
tumor during the course of observation in any of the mice.
Treatment group:
There were 14 mice each in the treatment arms of PC3 and LNCaP groups. Post
treatment one mouse/group was euthanized after 2 hrs, 24 hrs, 1 week and 2
weeks. The remaining mice (n=10) were sacrificed at 120 days.
Post injection, gross changes in tumor of greenish –grey color in the tumor and
overlying skin were noted by 2 hours (Picture -3), and became prominent by 6
hours. Ulceration and crusting of overlying skin were seen by 24 hours. The
scabs contracted and healed into scars by 23±5 days (Picture 2).
Gross tumor eradication was noticed in 17 (n=9 in PC3 and n=8 in LNCaP)
(Picture 4) mice at 120 days. Gross tumor recurrence was seen in 1 of PC3 and
2 of LNCaP mice. The recurrences were retreated successfully with a second
intratumoral injection. Microscopic tumor recurrence was noted in scar of 1
mouse of PC3 group on histopathology at 120 days.
Histopathological examination of the tumors from the mice euthanized after 2
hours post injection showed tumor degeneration and ischemic necrosis with
intravascular aggregation of red blood cells inside the tumor (Picture 5 and 6).
After 24 hours, areas of necrotic tissue with coagulative necrosis were noticed,
and the extent of cellular damage and tissue necrosis became more extensive.
After 1 and 2 weeks, severe degeneration with infiltration of fibroblasts and
granulation tissue was noted. Histopathology of the scars showed no evidence of
tumor cells, except in 1 specimen of PC3 group (Picture 7).
Discussion:
Prostate cancer represents a unique clinical problem with respect to treatment
options. Ninety percent of men will present with localized disease (12). For
localized prostate cancer, the current treatment paradigm is prostatectomy or
radiotherapy (external beam or bachytherapy). For men with advanced disease,
androgen therapy offers the best opportunity for long term survival. Given the
increased age at which many men present with prostate cancer and the slow
growing nature of this cancer, in many cases, the treatment options may have
equivalent morbidity in comparison to the cancer itself. Hence, less invasive
methods of treatment with fewer side effects would be very advantageous for
men presenting with localized disease. However, chemoablation of the prostate
cancer is not yet established (3).
There is much evidence in the literature to suggest that intratumoral treatment
with acetic acid for hepatocellular carcinoma has good results while being
systemically safe (10, 11). Also intra prostatic injection of acetic acid has been
used in humans to relieve the urinary obstruction secondary to benign prostatic
hyperplasia (4).
In this study significant tumor eradication was achieved with 25% acetic acid
injection into the PC3 and LNCaP tumors implanted in scid mice. Fifty percent
acetic acid has been used in humans safely. An initial pilot study was done to
find the acetic acid concentration which would achieve prostate tumor eradication
without causing any significant damage to peri tumoral tissues. Different acetic
concentrations of 5%, 10%, 25% and 50% were tried in 5 mice each. A direct
relation of peri tumoral tissue necrosis was observed with increasing acetic acid
concentration, with 5% causing the least and 50% causing maximal destruction
of surrounding tissues. A direct relationship of acetic acid concentration on tumor
recurrences was also noted, 5% group having more tumor recurrences while no
tumor recurrence was see in the group treated with 25% and 50%. Based on the
results of the pilot study 25% was selected as it caused tumor eradication with
minimal peri tumoral tissue damage.
Acetic acid should be correctly and carefully injected into the target site, since it
can degenerate and necrotize both the prostatic and adjunctive tissues. In this
study, complications such as skin ulceration and crust formation were observed
when acetic acid was injected at too large a volume ratio compared to the tumor
volume, or when the injection injured many capillaries to the skin since this tumor
was located subcutaneously. Pulling out the needle immediately after the end of
the injection might also have caused leakage to the surrounding tissue due to
backflow. In the clinical practice of local ethanol injection in patients with benign
prostate hypertrophy, the needle is removed after leaving it in the injection site
for one minute after injection. The reason is that if the needle is removed before
the ethanol permeates, there is a risk that the ethanol may flow back along the
path of the needle insertion (9).
Review of histopathology of the implanted tumors post injection showed ischemic
necrosis of the tumor cells starting immediately post injection. It translated to
considerable survival advantage in both prostate cancer cell line groups. Goya et
al. reported tumor regression with dehydrated ethanol injection (9) but multiple
intratumoral injections with high-dose ethanol were needed for tumor regression.
Repeated intratumoral injections may not be a desirable treatment modality for
human prostate cancer patients to achieve only tumor regression.
The present study showed that a single injection of acetic acid into tumors of
human prostate carcinoma cells in scid mice caused tumor eradication as well as
histological tumor cell injury and necrosis, suggesting that acetic acid injection
may be effective against human prostate cancer. This treatment option could
potentially be used in humans for treatment of early localized prostate cancer
and for non-operative management of symptoms in locally advanced cases. This
is the first report of a successful local chemo ablation for prostate cancer.
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Picture 1: A - LNCaP control group 8 weeks
B - PC3 control group at 5 weeks
Picture 2:
A & B- PC3 treatment group 6 days post injection
C & D - PC3 treatment group 2 weeks post
injection.
E & F - PC3 treated group 3-4 weeks post injection
Picture 3:
A- PC3 tumor at 5 weeks.
B - Tumor 2hours, post injection of acetic
acid.
C - Healed scar after 5 weeks.
Picture 4: PC3 treatment group at 120 days,
with scars.
Picture 5
Picture 6
Picture 7
A & B: Post treatment scar – no recurrence
C: PC3 post treatment scar (H&E) –tumor
recurrence surrounded by fibrosis.