Correlation of molecular and morphologic effects of thermoembolization in a swine model using mass spectrometry imaging

Article abstract of DOI:10.1002/jms.4477

Hepatocellular carcinoma is a growing worldwide problem with a high mortality rate. This malignancy does not respond well to chemotherapy, and most patients present late in their disease at which time surgery is no longer an option. Over the past three decades, minimally invasive methods have evolved to treat unresectable disease and prolong survival. Intra-arterial embolization techniques are used for large or multiple tumors but have distressingly high levels of local recurrence and can be costly to implement. A new method called thermoembolization was recently reported, which destroys target tissue by combining reactive exothermic chemistry with an extreme local change in pH and ischemia. Described herein are experiments performed using this technique in vivo in a swine model. A microcatheter was advanced under fluoroscopic guidance into a branch of the hepatic artery to deliver a targeted dose of dichloroacetyl chloride dissolved in ethiodized oil into the liver. The following day, the animals were imaged by computed tomography and euthanized. Assessing the reaction product distribution and establishing a correlation with the effects are important for understanding the effects. This presented a significant challenge, however, as the reagent used does not contain a chromophore and is not otherwise readily detectable. Mass spectrometry imaging was employed to determine spatial distribution in treated samples. Additional insights on the biology were obtained by correlating the results with histology, immunohistochemistry, and immunofluorescence. The results are encouraging and may lead to a therapy with less local recurrence and improved overall survival for patients with this disease.

Full text of DOI:10.1002/jms.4477

Received: 28 June 2019
DOI: 10.1002/jms.4477
Revised: 8 November 2019
Accepted: 15 November 2019
Journal of
MASS
S P E C I A L I S S U E ‐ R E S E A R C H A R T I C L E
SPECTROMETRY
Correlation of molecular and morphologic effects of
thermoembolization in a swine model using mass spectrometry
imaging
Chunxiao Guo1 Dodge L. Baluya1 Emily A. Thompson1 Elizabeth M. Whitley2
Erik N.K. Cressman1
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1 Department of Interventional Radiology, UT
Abstract
MD Anderson Cancer Center, Houston, Texas,
USA
Hepatocellular carcinoma is a growing worldwide problem with a high mortality rate.
This malignancy does not respond well to chemotherapy, and most patients present
late in their disease at which time surgery is no longer an option. Over the past three
decades, minimally invasive methods have evolved to treat unresectable disease and
prolong survival. Intra‐arterial embolization techniques are used for large or multiple
tumors but have distressingly high levels of local recurrence and can be costly to
implement. A new method called thermoembolization was recently reported, which
destroys target tissue by combining reactive exothermic chemistry with an extreme
local change in pH and ischemia. Described herein are experiments performed using
this technique in vivo in a swine model. A microcatheter was advanced under fluoro-
scopic guidance into a branch of the hepatic artery to deliver a targeted dose of
dichloroacetyl chloride dissolved in ethiodized oil into the liver. The following day,
the animals were imaged by computed tomography and euthanized. Assessing the
reaction product distribution and establishing a correlation with the effects are
important for understanding the effects. This presented a significant challenge, how-
ever, as the reagent used does not contain a chromophore and is not otherwise read-
ily detectable. Mass spectrometry imaging was employed to determine spatial
distribution in treated samples. Additional insights on the biology were obtained by
correlating the results with histology, immunohistochemistry, and immunofluores-
cence. The results are encouraging and may lead to a therapy with less local recur-
rence and improved overall survival for patients with this disease.
2 Department of Veterinary Medicine and
Surgery, UT MD Anderson Cancer Center,
Houston, Texas, USA
Correspondence
Erik N. K. Cressman, PhD, MD, FSIR,
Department of Interventional Radiology, UT
MD Anderson Cancer Center, 1400 Pressler
St. Unit 1471, Houston, TX 77030, USA.
Email: ecressman@mdanderson.org
Funding information
National Cancer Institute, Grant/Award Num-
bers: 1R01CA201127‐01A1 and P30
CA016672
KEYWORDS
ablation, dichloroacetate, embolization, liver cancer, thermochemistry
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INTRODUCTION
per year, and, unlike many cancers, the rate is increasing. Treatment
for HCC is inadequate for the large majority of cases, and mortality
Hepatocellular carcinoma (HCC) is a major global health issue for sev-
eral reasons. The incidence is high, estimated at over 850 000 cases
is correspondingly high.1 HCC frequently occurs in the setting of
underlying hepatic cirrhosis and presents at the intermediate to late
J Mass Spectrom. 2019;1–11.
wileyonlinelibrary.com/journal/jms
© 2019 John Wiley & Sons, Ltd.
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stages of disease. These realities compound an already difficult situa-
tion and limit therapy.
Liver transplantation is potentially curative, but only a small num-
ber of HCC patients qualify. There have been no substantial advances
in medical therapy since the very modest positive results with sorafe-
nib were first reported over a decade ago.2,3 Compliance with sorafe-
nib has proven a contentious issue because of side effects, further
limiting its impact on therapy.4 The checkpoint inhibitor nivolumab is
in early clinical trials, but there is no clear evidence to date that it will
provide a cure.5 As a result of these limitations, surgical resection and
locoregional therapies such as ablation or embolization play a promi-
nent role. Tumor size, multiplicity, location, and comorbidities are
among the factors that help to determine which of these treatment
options is most appropriate, but most patients present late in their dis-
ease and are not surgical candidates.6
Embolization methods are typically used when tumors are large or
multiple since, in these patients, neither surgical removal nor ablation
is feasible. In embolic therapies, a fluoroscopically guided catheter is
positioned in the artery just upstream of the target. Through the cath-
eter, physicians deliver drugs, particles, various liquid emulsions, or
combinations of these agents, resulting in ischemia and local tissue
death.7 Although there is a significant survival benefit, these treat-
ments are not curative. Examination of the explanted, diseased liver
after liver transplantation reveals that viable neoplastic tissue is found
in up to 90% of the previously treated liver specimens.8 Thus, there is
a clear need for improved therapies.
FIGURE 1 A, In situ thermoembolization reaction chemistry after
intravascular delivery of solution from a microcatheter.
Recently, a fundamentally new strategy for HCC therapy has been
developed. This approach, termed thermoembolization, is focused on
the local delivery of reactive compounds via image‐guided catheter
techniques to cause local tissue destruction.9,10 An example of such
a reaction is illustrated in Figure 1A, where hydrolysis of an electro-
philic reagent would release two equivalents of acid in situ and a con-
siderable amount of heat energy. A critical element of this approach is
mapping the distribution of the delivered reagents and/or the reaction
products within the target tissue in order to characterize and optimize
the therapeutic effects. Since many candidate compounds for this
application are not intrinsically fluorescent, mass spectrometry imag-
ing was evaluated to determine its potential to provide information
regarding spatial distribution and biological effects.11-13 Reported here
are the results of an investigation using an in vivo porcine model with
an acid chloride dissolved in ethiodized oil as the vehicle.
Dichloroacetyl chloride is strongly electrophilic and readily undergoes
hydrolysis in the presence of a nucleophile such as water. In the
process, the carboxylic acid is produced along with an equivalent of
hydrochloric acid, and a substantial amount of heat energy is released
(−96 kJ/Mole). Very localized, highly denaturing conditions are the
result. B, Thermoembolization causing rapid stasis in target vessel
distribution. A representative digital subtraction angiogram obtained
within 5 minutes after delivery shows X‐ray contrast in fine branching
vessels that supply unaffected areas on the left side of the image and
absence of contrast on the right in the embolized territory. Note
catheter tip (blue arrowhead) and abrupt cutoff of contrast with static
column (yellow arrow) and downstream absence of contrast in treated
area (green bracket territory) [Colour figure can be viewed at
wileyonlinelibrary.com]
were used directly as supplied. Contrast media (Iodixanol, Visipaque
320, GE Healthcare, Milwaukee, Wisconsin) was used directly as
supplied.
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MATERIALS AND METHODS
Reagents
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2.1
2.2
Animal care
Dichloroacetyl chloride (98%, Sigma Aldrich, St. Louis, Missouri) was
dissolved in ethiodized oil, which served as a radiopaque vehicle
(Lipiodol, Guerbet, Princeton NJ) at 2 mol/L and prepared immediately
before use. Reagents were used as supplied without further purifica-
tion. Sublimation matrices were 2,5‐dihydroxybenzoic acid (DHB)
and 9‐aminoacridine (9‐AA, Sigma Aldrich, St. Louis, Missouri) and
This study was performed with approval of the institutional animal
care and use committee. Three outbred swine were used. After induc-
tion using a combination of tiletamine hydrochloride and zolazepam
(50 mg/mL each at dosage of 4.4 mg/kg given intramuscularly, Telazol,
Zoetis US, Parsippany, NJ), the animals were intubated, and anesthesia
was maintained with 2% isoflurane supplemented with oxygen at up

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to 1.5 L/min as needed. During induction, buprenorphine was also
administered at 0.02 mg/kg given intramuscularly for analgesia, with
provision for additional postprocedure dosing as needed. Animals
were closely monitored before and after procedure with no significant
alterations in laboratory values as previously reported.10
approximately 60 to 80 seconds. After several minutes, a repeat
angiogram was performed in the treatment zone to document the
extent of the embolization and assess for stasis. Catheters and sheath
were then withdrawn from the access site, and 5 to 10 minutes of
manual pressure at the puncture site was used to achieve hemostasis.
The animals were then extubated and recovered from anesthesia.
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2.3
Embolization procedure
With the animals in the dorsal recumbent position, ultrasound guid-
ance was used to obtain needle access into the left common femoral
artery after which a wire was passed and ultimately exchanged for a
5F sheath (Cook Medical, Bloomington, IN). Through this access and
under fluoroscopic guidance, a reverse‐curve catheter was advanced
retrograde into the aorta and was used to engage the origin of the
celiac artery. A microcatheter and wire were then passed through
the base catheter and used to select a segmental artery in the liver.
Digital subtraction angiography was performed using iodinated con-
trast through the microcatheter to delineate anatomy and intermit-
tently thereafter to follow the course of the embolization, as well as
at the close of the procedure. To prevent vasospasm in the target area,
1% lidocaine (up to 5 mL) was slowly administered via the artery
immediately prior to treatment. The reactive thermoembolization solu-
tion was delivered through the catheter as a small bolus (400 μL)
surrounded on either end by a small column (150 μL) of ethiodized
oil, which was included in order to prevent contact of the reagent in
solution with blood or saline that could cause premature reaction
while still in the catheter. Delivery was accomplished over
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2.4
Macroscale imaging
The day following the treatment procedure, computed tomography
(CT) was performed using a 4‐slice scanner (General Electric Medical
Systems, Milwaukee, WI) with the animals under anesthesia. Data
were additionally processed using the Osirix MD (Pixmeo, Geneva,
Switzerland) to generate reformatted images.
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2.5
Euthanasia and tissue harvest
The day after the procedure and immediately after CT imaging, the
animals were euthanized by an overdose of phenytoin/pentobarbital
given intravenously. The liver was removed en bloc, and a CT scan
was obtained of the isolated organ. Small samples were snap frozen
in liquid nitrogen without embedding medium for mass spectrometry
imaging and serial frozen sections. Adjacent specimens were placed
in 10% neutral buffered formalin for histology.
FIGURE 2 Cross‐sectional imaging by noncontrast CT at 24‐hours post thermoembolization procedure. A, Axial image through the liver
demonstrating persistence of radiopaque ethiodized oil from the procedure, visualized as dense white stranding areas in an otherwise darker
field of surrounding liver. B, Reformatted pseudo‐color image of the liver showing arborization with embolic material appearing as bright linear
areas in the arterial branches. Note also the absence of the embolic material in the adjacent lobe, demonstrating controlled delivery to the target
region [Colour figure can be viewed at wileyonlinelibrary.com]

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2.6
Mass spectrometry imaging
2.7
Histology
Matrix was applied using the sublimation method with a Shimadzu IM
Layer (Shimadzu North America, Columbia, MD). Sampling was per-
formed using DHB for positive mode and 9‐AA for negative mode.
The number of laser shots used for these experiments was 300 shots
at 1 KHz using a laser pulse energy with an average of 25 μJ. For DHB
and 9‐AA, the laser was adjusted to 60% and 50%, respectively. The
mass range was from m/z 50 to 1200, and the instrument was calibrated
using peak signals from red phosphorus.
Sections of snap‐frozen liver were cut at five microns using a
cryomicrotome and stained with hematoxylin and eosin. Formalin‐
fixed sections of liver were processed routinely into paraffin blocks,
sectioned at five microns, and stained with hematoxylin and eosin.
Additional sections were used for immunohistochemical (IHC) and
immunofluorescence (IF) staining. Antibodies directed against
myeloperoxidase (ab9535, Abcam, Cambridge, MA) to detect neutro-
phils and cleaved caspase‐3 (CP2298, Biocare Medical, Pacheco, CA)
to detect apoptotic cells were used followed by secondary reagents,
including Bond Polymer Refine Detection system (DS9800, Leica, Buf-
falo Grove, IL) for chromogenic IHC and goat antirabbit Alexa Fluor
488 (ab150077, Abcam) for immunofluorescence staining. For IF,
polymerized actin of the cytoskeleton was detected using phalloidin
(Cytopainter Phalloidin‐iFluor 555, ab176756, Abcam), and nuclei
were detected using DAPI (Fluoroshield Mounting Medium with DAPI,
ab104139, Abcam). Stained sections were visualized using a Leica
DM2500 microscope with a Leica DFC495 camera and Leica Applica-
For MS/MS MSI analysis of taurodeoxycholic acid, parent ion at
m/z 498.295 was selected, and fragment ion at m/z 124.006 was
monitored at a collision trap voltage of 40 V. The laser shots were
500 shots per spot at 50% power.
Data were acquired using
a Waters Synapt G2 Si (Waters
Corporation, Milford, MA) at 60‐μm spot size with 100‐μm spacing.
Mass spectral data over tissue were averaged and imported into
Progenesis QI software for identification, and image reconstruction
was done with HD imaging 1.4.
FIGURE 3 Histology and fluorescence microscopy of thermoembolization. The procedure resulted in a targetoid zonal pattern of damage. The
most severe effects are centered on a portal triad (arrow in panel A). Note the loss of tissue architecture in the portal region and in the adjacent
hepatic lobule (panels A and B). Lysis of cells in the central region and infiltration of inflammatory cells (not identifiable at this magnification) result
in central loss of DAPI staining (asterisk in panel C) and accumulation of brightly DAPI‐positive nuclear material at the periphery of the lesion
(arrow in panel C). The line of demarcation between necrotic and viable tissue (arrow in panel D) is highlighted by the central loss and peripheral
retention of binding of the lectin phalloidin to polymerized actin proteins of the cytoskeleton. Note the bright red autofluorescence of
intravascular erythrocytes in the portal vein and at the periphery of the lesion (panel D). Porcine liver, hematoxylin and eosin (A), Phalloidin + DAPI
overlay (B), DAPI (C), and Phalloidin (D), bar = 500 microns [Colour figure can be viewed at wileyonlinelibrary.com]

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tion Suite v4.12 software or an Olympus BX41 with an Olympus U‐
RFL‐T source (Olympus, Center Valley, PA).
flow as a result of the treatment. Imaging by noncontrast CT the fol-
lowing day (Figure 2A) depicts the radiopaque ethiodized oil as dense
white areas in an otherwise darker background of surrounding liver. In
the reformatted false‐color image of the treated segment of the liver
(Figure 2B), the arborized vascular bed is readily appreciated because
of the contrast provided by the embolic material still present in the
vessels. The pattern of brighter branching lines of gradually smaller
caliber (embolized arteries) is clearly visible set against a background
of purple.
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3
RESULTS
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3.1
Macroscale imaging
A representative digital subtraction angiogram obtained within 5
minutes after completing the delivery of the reactive
thermoembolization solution (Figure 1B) shows X‐ray contrast mate-
rial flowing through and opacifying fine branching vessels that supply
nontarget, patent areas on the left side of the image. On the right side,
in the treated area however, there is no opacification of the vessels by
contrast material. This is consistent with disruption of antegrade blood
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3.2
Histology and mass spectrometry imaging
Histologic changes in treated regions of liver include well‐demarcated
areas of severe coagulative necrosis centered on portal triads contain-
ing a relatively large portal vein, hepatic artery, bile ductule, and
FIGURE 4 Histology, immunohistochemistry, and immunofluorescence demonstrating aspects of cell death in thermoembolization. There is
lethal and sublethal cell injury and the procedure incited an acute neutrophilic response and apoptosis within 24 hours. In the central region
(panel A), hepatic cords are disrupted and cytoplasmic and nuclear detail is lost, with cell lysis (asterisk) and loss of nuclear basophilia, consistent
with degradation of nucleic acids (arrow). More distant from the embolized hepatic artery (panel B), severely affected hepatocytes are lysed, with
release of cytoplasmic and nuclear contents (asterisk) or undergo coagulative necrosis (arrows). Neutrophils infiltrate into the injured areas (panel
C). At 24‐hour post ablation, scattered cells (arrows) peripheral to the zones of immediate cell death undergo programmed cell death (apoptosis),
demonstrating delayed and/or secondary injury from thermoembolization or inflammatory cell infiltrates, respectively. Porcine liver, hematoxylin,
and eosin (A and B) and immunostaining for myeloperoxidase (C) and cleaved caspase‐3 (D). In panel D, green = cleaved caspase‐3, blue = nucleic
acids, and red = phalloidin staining of polymerized actin of the cytoskeleton. Bar = 20 microns (A and B), 100 microns (C), and 500 microns (D)
[Colour figure can be viewed at wileyonlinelibrary.com]

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FIGURE 5 Geographic cell damage remote from site of direct contact with thermochemistry. Centrilobular hepatocytes in lobules adjacent to an
embolized artery of one pig are highly vacuolated and scattered apoptotic hepatocytes are present. These changes suggest a secondary effect on
the hepatic structures, possibly through hypoxia associated with reduced blood flow and/or through direct or indirect effects of embolization
agents. In a low magnification image (A), central regions of lobules adjacent to the embolized arteriole are pale. Arrow indicates area of higher
magnification (B), hepatocytes in the central vein (CV) region are swollen and pale, while hepatocytes near portal regions (P) have normal
morphology. Arrow indicates magnified area of CV central vein in (C), where some hepatocytes contain multiple, clear intracytoplasmic vacuoles or
have condensed, hypereosinophilic cytoplasm and nuclear morphology consistent with apoptosis (arrows). Magnification bars = 2000 microns (A),
200 microns (B), and 20 microns (C) [Colour figure can be viewed at wileyonlinelibrary.com]
extending across one or more hepatic lobules (Figures 3 and 4). In the
center of ablated regions, cells forming the portal arterial, venule, and
bile ductules are necrotic. In the lobular parenchyma, there is partial
retention of sinusoidal architecture with widespread hepatocyte
degeneration and death. In severely affected regions, binding of DAPI
to nuclear DNA and of the lectin phalloidin to polymerized actin of the
cytoskeleton is lost (Figure 3C,D, respectively). Peripheral to the
cleared, central area of necrosis is a zone of cellular and nuclear debris
and degenerating hepatocytes admixed with viable and degenerating
neutrophils. Apoptotic hepatocytes and neutrophils are present within
the zone of viable, injured tissue. In lobules adjacent to the embolized
artery, the centrilobular hepatocytes are vacuolated, and scattered
apoptotic hepatocytes are present (Figure 5). Necrosis was not
observed in untreated sections of liver or in control samples, where
ethiodized oil alone was administered.
fragment ions had a very similar distribution to the parent ion (Figure 7
, m/z 79.956, 106.980, and 124.006). A peak at m/z 496.324 [M − H]+
was tentatively assigned to a lysophosphatidylcholine (LPC, 16:0) with
the distribution most intense in a band surrounding the areas of
severe damage (Figure 8). Several additional extracted ion images
were noted with image correlation analysis that colocalized with the
LPC. HMDB (www.hmdb.ca) database search identified representative
peaks (m/z 575.48, 578.50, 738.43) tentatively assigned as diacylglyc-
erol DG(15:0/16:1), ceramide Cer(d18:0/d16:0), and phosphatidyleth-
anolamine PE(15:0/18:3). Similar findings were observed in samples
from two additional animals (Figures S1 and S2).
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4
DISCUSSION
The frozen sections corresponding to the mass spectrometry sam-
ples likewise contain well‐demarcated regions of acute necrosis
rimmed by neutrophilic inflammation and injured hepatic parenchyma
(Figure 6A‐F). Negative ion‐mode mass spectrometry images illustrate
the distribution of common cell lipids that establish the correspon-
dence to the adjacent stained section and distribution of intact cells.
For example, m/z peaks at 885.521 corresponding to phos-
phatidylinositol (PI 38:4) and 281.2393 corresponding to oleic acid
(OA) are homogeneously distributed throughout the data set with a
notable exception. These peaks are absent in the areas of greatest
damage visible on the frozen section (Figure 6G‐L). The peak at m/z
126.9014 corresponding to iodide aligns well, situated centrally within
these same areas of damage. In addition, peaks at m/z 448.290,
496.257, and 498.273 were also identified in these areas and
corresponded to approximately the same distribution. These peaks
were tentatively assigned to glyco (cheno)deoxycholate [M‐H]−,
taurocholate [M‐H2O‐H]−, and taurodeoxycholate [M − H]−, respec-
tively, based on accurate mass. MS/MS was obtained on an adjacent
section and supported the assignment for taurodeoxycholate. All three
Mass spectrometry imaging of this new method provided an interest-
ing snapshot of hepatic physiology and pathology at the molecular
level. The thermoembolization procedure as tested proved very effec-
tive, both in achieving rapid intravascular stasis and in causing
targeted regional cell death. Cross‐sectional imaging using CT demon-
strated that the embolic reagent solution did not wash out of the tis-
sue over the course of 24 hours. Both the degree of damage and the
physical extent of the damage observed on histologic examination
suggest that eventual recanalization and restoration of flow in the
treated areas (seen with some other embolic agents) would be highly
unlikely. That being the case, it was anticipated that there would be
evidence at MSI of the embolic solution and exothermic reaction
occurring in situ. The finding of the m/z peak at 126.901 assigned to
iodide supports this idea, since the ethiodized oil that was used as a
carrier for DCACl contains substantial amounts of iodine. The use of
iodized oils to provide a slow release source of iodide for goiter is
well‐known, providing ample precedent for the presence of iodide.14
The pattern of distribution across the specimen for this peak is consis-
tent with the arterial delivery method.

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FIGURE 6 Histology and mass spectrometry imaging of thermoembolization (negative mode, 9‐AA matrix). A, Frozen section H&E of adjacent
section as a reference. B, Phosphatidyl Inositol or PI 38:4, 885.521 [M − H]−, (C) oleic acid or OA, 281.2393 [M − H]−, (D) iodide, 126.9014 [M –
H], (E) overlay of PI and iodide, (F) overlay of iodide with H&E. Damaged areas correlate with areas with iodide present. G, Taurodeoxycholate or
TDC, at m/z 498.2723 [M − H]−, (H) overlay of TDC with PI 38:4, (I) MS of taurocholic acid at m/z 496.2576 [M − H2OH]−, (J) Glyco (cheno)
deoxycholate) at m/z 448.2901 [M − H]−, (K) Overlay of Glyco (cheno)deoxycholate) with PI, (L) Combined overlay of PI 38:4, TDC, and glyco
(cheno)deoxycholate. Compare localization with D‐F depicting distribution of iodide [Colour figure can be viewed at wileyonlinelibrary.com]
The normally ubiquitous lipids PI and OA were both absent in the
areas of most severe damage (Figure 7B,C). A possible explanation is
that the combination of heat and acidity from the reaction could
potentially cause breakdown if they proved unstable to the local
milieu. In the damaged areas, three additional, unexpected m/z peaks
were identified. These were tentatively assigned based on accurate
mass data to glyco (cheno)deoxycholate, taurocholate, and
taurodeoxycholate, with further supportive MS/MS data in the last
case. These compounds are naturally occurring bile salts that are nor-
mal constituents of liver tissue.15,16 Bile salt synthesis and secretion
by the liver normally fluctuate with oral intake and are reduced during
fasting.17,18 Animals were fasted overnight for each of the two
anesthetic/imaging events separated by 24 hours in accordance with
institutional policy. Taking these factors together, we speculate that
the bile salt distributions reflect this physiology. One possible result
would be that the bile salts detected in the lesions would have been
present at the time of the thermoembolization and were retained in
the necrotic tissues because of the acute loss of blood and bile flow.
The absence of m/z peaks for these bile salts in the viable, nontreated
liver may reflect the combination of further reduction in bile salt

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FIGURE 7 Potential biomarker candidates for ischemia. Images of representative m/z signals noted with increased intensity at perimeter of
damaged areas with assignments as follows. (A) Lysophosphatidylcholine LPC (16:0) m/z 496.324 [M + H]+, (B) diacylglycerol DG (33:4) m/z
575.484 [M + H]+, (C) ceramide Cer(t18:0/16:0) m/z 578.503 [M + Na]+, (D) phosphatidylethanolamine PE (33:3) m/z 738.432 [M + K]+, (E) larger
view of A with yellow arrows highlighting the perimeter pattern. (F) H&E section [Colour figure can be viewed at wileyonlinelibrary.com]
production combined with reduced enterohepatic circulation associ-
ated with preanesthetic fasting.
From a histologic perspective, the damage was clearly centered on
vascular structures as might be predicted based on the method of
delivery. Coagulation necrosis reached well beyond the arterioles
and involved the adjacent portal structures and deep into the regional
hepatic parenchyma. Indeed, directly damaged areas spanned as much
as several hundred microns in radius and frequently crossed the col-
lagenous interlobular septae. This distance approaches that seen with
radioembolization (90Y, a beta emitter) but without the need for a
radioisotope.20
An interesting incidental finding in positive mode was the ring‐like
distribution of lipids in Figure 8 surrounding the most severe injury.
This was interesting in another respect since the putative LPC at m/z
496.324 [M + H]+ was recently posited to be a biomarker of ischemia
in renal tissues.19 If this finding is generalized in different tissues, it
would suggest a pattern of ischemia in the perimeter of the areas with
the most extreme damage. Moreover, additional lipids were identified
with a similar pattern of distribution. While the data are preliminary in
nature, when taken together, this raises an interesting possibility that
these lipids may be biomarkers of ischemia.
The extent of damage caused by thermoembolization also raises
some other physiologic and pharmacologic points. First, the zone of
cell death is substantially larger than the distribution of the peak

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FIGURE 8 (A) MS/MS spectra of taurodeoxycholic acid standard at m/z 498.29 [M − H]− producing fragment ions at m/z 79.957, 106.982, and
124.010. (B‐D) Extracted ion images of fragment ions as follows: (B) m/z 79, (C) m/z 106, and (D) m/z 124 from the MS/MS imaging analysis for
taurodeoxycholate [Colour figure can be viewed at wileyonlinelibrary.com]
assigned to iodide as indicated by MSI. This suggests at least two pos-
sible injurious effects directly related to thermoembolization are oper-
ative in areas that are clearly devitalized but well beyond the
geographic areas that have detectable amounts of iodide. One effect
is the heat from the exothermic hydrolysis that is predicted by itself
to result in tissue coagulation. A second likely effect is due to diffusion
of acid away from the vessel and out into the adjacent tissues. Other,
secondary mechanisms such as ischemia as noted above from vascular
embolization and cytotoxic effects of the cellular inflammatory
response are also likely factors partly accounting for observed tissue
damage. Perhaps most likely, the outcome reflects a combination of
these effects in a synergistic manner, although the relative contribu-
tion of each of these effects is not known.
blood supply in liver. Recent studies with hyperpolarized imaging in
embolized tumors have provided new evidence that portal flow and
autophagy may in fact contribute to cell survival after
chemoembolization.21 While there is only preliminary evidence
supporting this concept, destruction of the portal venules observed in
the current studies may prove to be beneficial by preventing the deliv-
ery of oxygen and nutrients to the embolization zone.
The observation of extensive centrilobular vacuolization and apo-
ptosis in the regions surrounding the most extreme areas of ablation
but with few signs of thrombosis in these lobules bears further com-
ment. This change was identified in numerous lobules that did not
have zones of coagulation necrosis in the immediate proximity and
were located several lobules distant from the severely affected artery.
In this context, it is well‐known that the centrilobular region is most
prone to ischemic insults. Results indicate that damage to the treated
arteries was extreme. This implies that the arterial blood supply to
downstream regions would be interrupted. Taking these into consider-
ation, this pattern of injury may represent downstream ischemic
effects of thermoembolization. Survival of hepatocytes in affected
lobules may be attributable to the presence of nutrients and oxygen
in portal blood from incomplete destruction of portal structures or
from collateral circulation.
The extensive damage to the portal venous system and other struc-
tures in the portal triad may have implications beyond local tissue dam-
age. It is possible that the historically high rates of incomplete treatment
in conventional embolization procedures may be related to some
degree by their limited impact on the portal blood supply. Although
the portal blood has a lower oxygen content than arterial blood, relative
to tumor tissue, it is still quite oxygenated and is also very nutrient rich.
The importance of this may be underappreciated because of the
established literature on the general dominance of the arterial tumor

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SPECTROMETRY
Given the relatively short time period of 24 hours after
ACKNOWLEDGEMENTS
thermoembolization and before euthanasia, the full extent of tissue
damage and the immune response resulting from thermoembolization
is not expected to be apparent in these samples. At 24 hours after
thermoembolization, a robust infiltrate of neutrophils is present at
the periphery of the severely injured zone and represents a host
response to the nucleic acids, proteins, and ATP released during
necrosis. The release of cellular contents serves as damage‐associated
molecular patterns or “danger signals” to the host that result in the ini-
tiation of a noninfectious inflammatory response.22,23 The neutrophils
attracted to the embolization site help create a sterile proinflammatory
environment into which it would be expected that classically activated
(M1) macrophages or Kupffer cells will be recruited. In addition, in an
incompletely ablated tumor, the inflammatory response initiated by
embolization might be able to reprogram tumor‐associated alterna-
tively activated (M2) macrophages to a proinflammatory phenotype
and thus carry out tumoricidal activities and initiate an adaptive
immune response.24,25
This work was supported by NIH 1R01CA201127‐01A1, MD Ander-
son Cancer Center Institutional Research Grant program, NIH CCSG
P30 CA016672, and The James B. and Lois R. Archer Foundation,
the E.L. Wiegand Foundation, and the J.S. Dunn Foundation. The
authors thank Amanda McWatters and Katherine Dixon for technical
and administrative support and the personnel in the Department of
Veterinary Medicine and Surgery for animal care and histologic
processing.
ORCID
Erik N.K. Cressman https://orcid.org/0000-0002-8671-9862
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