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Case Report: Microfragmented Adipose Tissue Drug Delivery in Canine
Mesothelioma: A Case Report on Safety, Feasibility, and Clinical Findings
ArticleinFrontiers in Veterinary Science · January 2021
DOI: 10.3389/fvets.2020.585427
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CASE REPORT
published: 14 January 2021
doi: 10.3389/fvets.2020.585427
Frontiers in Veterinary Science | www.frontiersin.org 1January 2021 | Volume 7 | Article 585427
Edited by:
David Bruyette,
Anivive Lifesciences, United States
Reviewed by:
Elena De Felice,
University of Camerino, Italy
Chad M. Johannes,
Iowa State University, United States
*Correspondence:
Francesco Petrella
francesco.petrella@ieo.it;
francesco.petrella@unimi.it
Specialty section:
This article was submitted to
Comparative and Clinical Medicine,
a section of the journal
Frontiers in Veterinary Science
Received: 20 July 2020
Accepted: 04 December 2020
Published: 14 January 2021
Citation:
Zeira O, Ghezzi E, Pettinari L, Re V,
Lupi DM, Benali SL, Borgonovo S,
Alessandri G, Petrella F, Paroni R,
Dei Cas M, Tremolada C, Coccè V
and Pessina A (2021) Case Report:
Microfragmented Adipose Tissue
Drug Delivery in Canine
Mesothelioma: A Case Report on
Safety, Feasibility, and Clinical
Findings. Front. Vet. Sci. 7:585427.
doi: 10.3389/fvets.2020.585427
Case Report: Microfragmented
Adipose Tissue Drug Delivery in
Canine Mesothelioma: A Case Report
on Safety, Feasibility, and Clinical
Findings
Offer Zeira 1, Erika Ghezzi 1, Letizia Pettinari 1, Valentina Re 1, Davide M. Lupi 1,
Silvia L. Benali 2, Simone Borgonovo 3, Giulio Alessandri 4,5 , Francesco Petrella 6,7
*,
Rita Paroni 5,8 , Michele Dei Cas 8, Carlo Tremolada 9, Valentina Coccè 5and
Augusto Pessina 5
1Department of Stem Cells and Regenerative Medicine, San Michele Veterinary Hospital, Tavazzano con Villavesco, Italy,
2Laboratorio La Vallonea, Milan, Italy, 3Clinica Veterinaria Crema, Crema, Italy, 4Department of Cerebrovascular Diseases,
Istituto di Ricovero e Cura a Carattere Scientifico, Besta Neurological Institute, Milan, Italy, 5Centro di Ricerca Coordinato
StaMeTec, Department of Biomedical, Surgical and Dental Sciences, University of Milan, Milan, Italy, 6Department of Stem
Cells and Regenerative Medicine, Istituto di Ricovero e Cura a Carattere Scientifico, European Institute of Oncology, Milan,
Italy, 7Centro di Ricerca Coordinato StaMeTec, Department of Oncology and Emato-Oncology, University of Milan, Milan,
Italy, 8Department of Health Sciences, Università Degli Studi di Milano, Milan, Italy, 9Department of Stem Cells and
Regenerative Medicine, Istituto Image, Milan, Italy
Mesothelioma is a rare lethal tumor of dogs and humans involving cavities of the
body. Dogs are considered a model for new drugs and therapeutic methods since
they present spontaneous diseases similar to humans. Microfragmented adipose
tissue (MFAT) uploaded by paclitaxel (PTX) is a drug delivery medium providing
slow release of chemotherapic drugs. A dog affected by pleural, pericardial, and
peritoneal mesothelioma was treated by 17 intracavitary ultrasound-guided injections
of MFAT-PTX over 22 months. A long-lasting improvement of general conditions was
observed, treatment was well-tolerated, and no toxicity or hypersensitivity was reported.
Pharmacokinetic (PK) data indicated low drug localization in the circulatory system
and a tendency to enter or remain in the extravascular compartments of the body.
Indeed, low levels of free-circulating drugs for a short time produced low toxicity,
whereas, a higher intracavitary PTX concentration can have major pharmacological
efficacy. To our knowledge, this is the first time that mesothelioma has been treated
using such a procedure, and this should be considered as a novel therapeutic
approach. The low systemic absorption suggests the possible role of MFAT-PTX for
loco-regional/intratumoral therapy also useful in other types of tumors, and further
investigation is warranted.
Keywords: mesothelioma, paclitaxel, adipose tissue, dog, drug delivery
BACKGROUND
Mesothelioma is a rare neoplasm of dogs and humans affecting the lining epithelial cells of
the coelomic cavities of the body. It is a chemoresistant tumor and actually has no effective
therapeutic strategies. In human medicine, around 80% of this tumor occurs in men, median
survival is 8–18 months, and the survival rate is <12% in the 5 years after diagnosis.
Zeira et al. Case Report: Loco-Regional Treatment of Dog’s Mesothelioma
In veterinary medicine, no sex predilection is reported. Median
survival for untreated dogs with mesothelioma is difficult to
assess since they are often euthanatized following diagnosis.
Various studies of treated dogs (surgery, intracavitary, and
intravenous chemotherapy) report survival time from 2 to 13
months (1). In both humans and dogs, the tumor may involve
cavities such as thorax, abdomen, pericardial sac, and the vaginal
tunics of the scrotum.
The domestic dog is considered to be an important animal
model for the evaluation of new drugs and therapeutic
methods since it presents spontaneous diseases very much
similar to human oncology (2,3), including mesothelioma.
Microfragmented adipose tissue (MFAT) is a potential
drug delivery medium that may provide a slow release of
chemotherapic drugs contiguous to the tumor (4). MFAT
consists of stromal vascular fraction, pericytes, and adipose
stromal stem cells, and it is known to have trophic, mitogenic,
anti-scarring, anti-apoptotic, immunomodulatory (5), and
antimicrobial actions produced by a large number of bioactive
elements, growth factors, and cytokines (5–7).
In order to obtain MFAT, we employed a commercially
available, enzyme-free technology (Lipogems) able to harvest
micro-fragmented fat preparation. This technology reduces the
size of the adipose tissue clusters by means of mild mechanical
forces while eliminating pro-inflammatory oil and blood residue.
The technique is gentle and provides micro-fragmented fat
in a short time (15–20′) without expansion and/or enzymatic
treatment (8,9). Paclitaxel (PTX, Taxol R
) is an anticancer
chemotherapy drug that isclassified as a “plant alkaloid” and
is used in various types of solid tumors in human medicine.
Although the hypersensitivity and side effect related to the
toxicity of paclitaxel and cosolvents are well-known (10),
numerous studies have suggested the efficacy of paclitaxel in
different types of tumors using both systemic and subcutaneous
injection (11–14). Whereas, intracavitary chemotherapy in
veterinary mesothelioma was described using platinum-based
drugs (15), paclitaxel is not considered the therapy of choice for
mesothelioma. In agreement with new approaches oriented to
verify and validate new paclitaxel formulations, the aim of our
study is to verify the safety, feasibility, and efficacy of intracavitary
administration of paclitaxel-loaded microfragmented adipose
tissue in mesothelioma in dogs.
CASE PRESENTATION
Patient
A 6-year-old, mixed-breed, 24-kg, neutered dog, with a 2-
month history of a progressive weakness, loss of appetite,
productive cough, abdominal distension, and difficulty in
breathing, was selected for the treatment. Complete blood
count (CBC) and biochemical tests were assessed. Thoracic
radiographs detected severe pleural effusion and bilateral middle
and caudal lung lobe collapse. Abdominal radiographs and
ultrasound also evidenced severe effusion. Serosanguinous fluid
was removed from the pleural and abdominal spaces, underwent
cytological examination, and showed high malignant characters
(anisocytosis and anisokaryosis). Ultrasound-guided biopsies
were taken from the pleura, pericardium, and peritoneum,
and histological and immunohistochemical diagnoses were
performed; the final diagnosis was of mesothelioma.
Paclitaxel-Loaded Adipose Tissue
(MFAT-PTX) Preparation
Autologous adipose tissue, obtained by lipoaspirate from
the dog’s lumbar flanks, was microfragmented by using a
microfragmentation device (Lipogems, Italy) as previously
described (16). This procedure allows minimal manipulation
without the use of enzymatic procedures, and microfragmented
samples can be also cryopreserved or used as a scaffold
for paclitaxel (MFAT-PTX). For our study, fresh aliquots of
microfragmented adipose tissue were loaded with PTX by adding
the drug at a concentration of 1 mg/ml and stirring the mixture
for 30 min before use as previously reported (4).
Chemotherapeutic Protocol
Treatment protocol consisted of ultrasound-guided
administration into the abdominal and thoracic cavity of
7 ml of MFAT-PTX (1 mg/ml) corresponding to 0.29 mg/kg
(0.35 mg/m2) of free drug. First administration (T0) was done
15 days after presentation. Over the course of 22 months,
the dog underwent 17 treatments (both intrathoracic and
intra-abdominal), with an average of a treatment every 38 days
(shortest interval 14 days, longest 70 days).
The procedure is initiated by drainage of the abdomen and
chest from the exudate fluid followed by injection of 3 ml
of MFAT-PTX intraperitoneally +2 ml, respectively, into the
right and left pleural spaces (Figure 1). Clinical outcomes were
monitored at the time of each treatment and documented by
thorax radiographs, abdominal ultrasound, clinical examination,
and blood counts. Special attention was given to eventual
adverse effects.
Pharmacokinetics (PK) Study
The minimal parameters of PK [Cmax, Tmax , T1/2, volume of
distribution (Vd), and area under curve (AUC)] were evaluated
by checking the amount of drug in the blood 30 min and 2,
4, and 8 h after the first treatment of the MFAT-PTX. At the
10th treatment (T10), the residual amount of the drug was
also measured in the pleura and pericardium biopsies. The
amount of PTX was evaluated by liquid chromatography-coupled
mass spectrometry (LC-MS/MS) analysis as already reported (4).
Briefly, blood (100 µL) and tissues (about 50 mg) were added
in internal standard (25 µl of paclitaxel D5 10 µg/ml) and PTX
was extracted by 1 mL of a mixture methanol/isopropanol (6:4,
v:v). Dry extracts were redissolved with 200 µl of acetonitrile
and 10 µl injected in LC-MS/MS. The details for instrumental
conditions were already described elsewhere and kept essentially
unaltered (4).
Clinical Assessment
No major short- or long-term adverse effects were registered.
Complete blood counts and biochemistry performed each month
did not show abnormalities. In particular, during the whole
treatment leukocytes and platelet counts in the peripheral blood
Frontiers in Veterinary Science | www.frontiersin.org 2January 2021 | Volume 7 | Article 585427
Zeira et al. Case Report: Loco-Regional Treatment of Dog’s Mesothelioma
FIGURE 1 | Lipoaspirate collection and ultrasound-guided intrathoracic treatment with MFATPTX. (A) Lipoaspirate collection from the dog’s lumbar flanks. (B)
Procedure of treatment that was initiated by drainage of abdomen and chest from the exudate fluid followed by the injection of 3ml of MFAT-PTX intraperitoneally +
2 ml, respectively, into the right and the left pleural spaces. (C) Ultra sound view.
indicated constantly normal values, suggesting an absence of
systemic myelotoxicity. All phases of the treatments were feasible.
In order to avoid iatrogenic damage to the internal organs, major
attention was given to the intracavity administration when a low
amount of effusion was present.
The dog presented rapid improvement in its general
conditions after each exudate fluid drainage, followed by the
intracavitary administration. This effect lasts for an average
period of 30–40 days.
Notably, in the first 15 months of treatment the intervals
between treatments were longer (average 52 days) while in the last
7 months the intervals between treatments were shorter (average
24 days).
The patient presented good clinical conditions during
most of this period. During the final few days before each
treatment, exudate formation became visible, and the clinical
conditions worsened.
Evaluation of the clinical status was based on the periodical
veterinarian controls in which exudate fluid formation was
assessed by ultrasound, radiographs, and computed tomography
scan (CT) together with breathing quality. Both parameters
presented clear amelioration. Notably, the abdominal exudate
production completely ceased after the fourth treatment. In
addition, the owner’s evaluation was taken into consideration,
as they had knowledge of the dog’s “normal” vs. “abnormal”
behavior. The owner reported a good quality of life. The
dog was able to play with other dogs, go up the stairs, and
coughed rarely, breathed normally most of the time, and had
a good appetite.
Imaging Diagnostic Assessment
Post-treatment thoracic radiographs, performed once a month
in the first 6 months, showed progressive reduction of the
severe pleural effusion (Figure 2). The following radiographs,
every 3 months, evidenced only mild differences in the thoracic
effusion quantity while in the abdominal cavity no liquid could be
detected. The first thorax and abdominal CT scan, pre- and post-
contrast medium, performed 8 months after the initial treatment
evidenced mild to moderate pleural effusion, rare abdominal
effusion, pleura and peritoneum reactivity, and loco-regional
lymphadenopathy. A control CT after 12 months showed only
mild difference (Figure 2).
Pathological Assessment
The patient was euthanized after 22 months due to worsening of
his clinical conditions and underwent a complete post-mortem
exam. Macroscopically all the pleural surfaces presented isles
of yellowish fatty material, identified as MFAT-PTX (last
administration 2 weeks previously) (Figure 3). Cytological
smears of pleural and peritoneal effusion were evaluated,
presenting overlapping findings: hematic background with
scattered eosinophilic material, good cellularity, and mixed
cellular population; and presence of numerous cells in
clusters of small/medium size, having variable cytoplasm
nucleus/cytoplasm ratios, slightly basophilic cytoplasm with
occasional vacuolization, round/oval central or paracentral
nucleus with coarse chromatin, and prominent central nucleolus.
The cells present moderate to severe malignancy characters,
Frontiers in Veterinary Science | www.frontiersin.org 3January 2021 | Volume 7 | Article 585427
Zeira et al. Case Report: Loco-Regional Treatment of Dog’s Mesothelioma
FIGURE 2 | Radiography and computed tomography scan (CT) along the treatment. The figure reports the post-treatment thoracic radiographs (Rx) performed once
a month in the first 6 months. They showed progressive reduction of the severe pleural effusion. The computed tomography scan (CT) shows the first CT scan 8
months after first treatment. Transversal view of the soft tissue window, and post-contrast right side effusion. The control 12 months after the first CT scan shows a
bilateral pleural effusion mildly increased.
including anisocytosis and anisokaryosis and presence of
occasional leukocytes.
A biopsy of pericardium was fixed in 10% buffered formalin,
embedded in paraffin, and stained routinely with hematoxylin
and eosin. Immunohistochemistry was performed on paraffin
sections (30 µm) placed on Superfrost Plus slides (Superfrost R
Plus) with an automated immunostainer (Discovery Ultra-
Roche). The primary antibody was a monoclonal anti-mouse
mesothelial cell clone HBME-1 (Dako cod M3505) diluted 1:100
and incubated at room temperature for 20 min.
Histopathological sections of pericardium and pleura were
examined with focal evidence of a neoplastic cellular component
within the pleura engorging dilated vessels. Cells were round
to polyhedral and arranged in small groups or micropapillae.
The nucleus had a cytoplasmic ratio that was intermediate.
The cytoplasm moderate and eosinophilic, and the nucleus was
round to oval with 1–2 nucleoli. Anisokaryosis was marked and
anisocytosis moderate. Mitosis were 0–2 in 10 high-power fields
(400×). Immunohistochemistry for the HBME–mesothelioma
marker confirmed the mesothelial origin of the cells (Figure 3).
Pharmacokinetics (PK) Assessment
After treatment with MFAT-PTX, no drug was detected in the
blood at 30 min. At 2, 4, and 8h, the amounts detected in plasma
were 28.8, 19.25, and 4.89 ng/ml, respectively (Figure 4). Cmax
was 28.8 ng/ml, Tmax =2 h, and the half-time (T1/2)=5 h.
Based on the dose of PTX injected and the plasmatic Cmax , we
estimate an apparent volume of distribution at 2 h (Vd2h) of
243 liters. The calculation of plasmatic AUC 1–8 h demonstrated
that MFAT-PTX treatment makes available the drug in plasma
with a value of 117 ng∗h/ml. The residual amount in pleura
and pericardium measured at 30 days after the 10◦treatment by
HPLC suggested the presence of 3.6 and 13.3 ng/g, respectively,
which, by considering the weight of these tissues, can be evaluated
as a residual drug of about 2.16 µg in the pleura and 3.99 µg in
the pericardium.
DISCUSSION
Mesothelioma is a fatal disease in both dogs and humans,
and new effective therapeutic strategies are needed (17).
Our data suggest that localized delivery of microfragmented
adipose tissue (MFAT) uploaded by paclitaxel (MFAT-PTX)
directly into the peritoneal and thoracic cavity is feasible.
Administration in average intervals of 38 days was well-tolerated
in the dog, and no unique toxicity or hypersensitivity was
noted. This result is of particular interest. Due to the low
aqueous solubility of paclitaxel, Taxol R
formulations include
Frontiers in Veterinary Science | www.frontiersin.org 4January 2021 | Volume 7 | Article 585427
Zeira et al. Case Report: Loco-Regional Treatment of Dog’s Mesothelioma
FIGURE 3 | Macroscopic and histological analysis of pleura. (A) The macroscopic analysis shows the presence on the pleural surfaces isles of yellowish fatty material,
identified as MFAT-PTX (last administration 2 weeks previously). (B,C) Histological section of pleura (hematoxylin and eosin stain) shows neoplastic cells within the
lumen of dilated vessel. Immunohistochemistry for the HBME–mesothelioma marker shows moderate to intense membranous immunolabeling of the tumor cells with
apical membrane accentuation.
FIGURE 4 | Plasmatic concentration of paclitaxel (PTX). The figure shows the
plasmatic concentration of PTX after the first treatment with MFAT-PTX as
reported in the chemotherapeutic protocol. The amount of PTX was checked
in the blood after 30 min and 2, 4, and 8 h.
Cremophor R
(polyoxyethylated castor oil) and ethanol as an
excipient. Such formulations overcome poor solubilization
of paclitaxel for parenteral use. However, Cremophor R
-
induced complement activation is believed to be the cause
of common hypersensitivity reactions related to Taxol R
use
in humans and other species (10,18,19). The absence of
hypersensitivity in our case may be explained by various
ways. MFAT is known to have strong immunomodulation
activity (9). Another possible explanation is the long
intervals between treatments together with the slow release of
low doses.
The MFAT-PTX scheme of treatment seems to be able to
produce a local, rather long-term, antineoplastic effect without
any systemic myelotoxicity. A CT control scan after 12 months
showed no major difference in the effusion quantity, pleural and
peritoneal reactivity, and lymphadenopathy, which indicates a
reduction in tumor progression. The long-lasting improvement
in the dog clinical conditions was regularly reported from both
veterinarian and owner and is probably due to the slow release
of paclitaxel from the microfragmented adipose tissue in the
thorax and abdomen, as already reported (4). As far as the
thoracic cavity is concerned, from our data in the first 15
months of treatment, the intervals between local administrations
were longer and became progressively shorter in the following
months (from average of 52–24 days). The need to shorten the
intervals in the last months of treatment was due to a slow and
progressive increase in the thoracic effusion. On the other hand,
in the peritoneal space, effusion quantity was very much reduced
within 6 months after the initial treatment and remained such
until the last treatment. This observation is important since the
dose of paclitaxel and MFAT volume remained the same in all
treatments. It may find an explanation by assuming that the
tumor developed some grade of resistance, but it does not explain
why the peritoneal compartment behaved in a different way,
Frontiers in Veterinary Science | www.frontiersin.org 5January 2021 | Volume 7 | Article 585427
Zeira et al. Case Report: Loco-Regional Treatment of Dog’s Mesothelioma
maintaining a very low level of effusion during the whole period.
In order to answer this query, a much larger number of patients
are warranted.
The PK data showed a very high value of Vd indicating that,
after treatment, the drug localization in the circulatory system is
low and that the drug has a propensity to enter or remain in the
extravascular compartments of the body. This is compatible with
the chemical structure of PTX, which is a lipophilic molecule. It
is also a consequence of the loco-regional treatment with MFAT-
PTX resulting in a more significant distribution of PTX into the
areas with higher lipid density.
As known, this parameter together with the above reported
gives an index of the systemic drug exposure. The low value
is predictive of low systemic toxicity due to the loco-regional
treatment producing low levels of free-circulating drug for
a short time, whereas, locally, PTX concentration can have
pharmacological efficacy. This is also confirmed by the low
modulation of blood cell counts during the treatment and by the
presence of significant residual amounts of PTX found in both
pleura and pericardium 1 month after treatment.
Even though our experience with this therapeutic procedure
includes more than one patient, we have decided to limit our
report to this single case due to its very intense follow-up and
detailed investigation from diagnosis to post-mortem assessment.
To our knowledge, this is the first time that mesothelioma
is treated using such a procedure in a dog and should be
considered as a novel therapeutic approach for mesothelioma
treatment. Furthermore, the lack of systemic absorption after
intra-abdominal and intrathoracic administration suggests a
possible role of MFAT-PTX for intratumoral therapy. Such a
procedure may also be useful in other types of tumors, and
further investigation is warranted.
We have used this new procedure in 2 other cases of
mesothelioma. Unfortunately, both cases were presented to
our hospital in a rather advanced clinical status and died
within 1 month for cardiopulmonary failure. However, the same
procedure was applied to other 2 patients with tumors such as
spleen hemangiosarcoma and ovarian carcinoma with diffuse
abdominal involvement. At the present time, results are similar to
our mesothelioma case by means of no major short- or long-term
adverse effects and overall survival time.
The lack of complications in the dog should be taken
into account when considering this treatment in other species,
including man. The study of spontaneous, naturally occurring
tumors in dogs is a model that provides a valuable role in
developing potentially successful, innovative treatment regimens
for translational medicine, facilitating the transfer of knowledge
from the “bench” to the “bedside.”
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation.
ETHICS STATEMENT
The procedures involving MFAT-PTX were performed in
accordance with the guidelines defined by the Italian Presidency
of the Council of Ministers and following the guidelines
published by the General Directory of Animal Health and
Veterinary drugs of the Italian Ministry of Health. The
owners considered euthanasia for their dog but accepted our
proposed treatment as a last possibility. They were thoroughly
informed about the entire procedure and signed a formal
agreement with the San Michele Veterinary Hospital in
acceptance of both anesthesia and therapy. They also accepted
that their dog would undergo post-mortem examination. The
veterinary hospital followed guidelines established for Good
Clinical Practice.
AUTHOR CONTRIBUTIONS
OZ, LP, EG, VR, DL, SLB, and SB: medical diagnosis,
management of case, and collection of data. RP, MD, and CT:
laboratory analysis. AP, EG, VC, GA, and FP: collection of data,
writing, and editing of manuscript. AP, VC, and FP: review
of final submission. All authors contributed to the article and
approved the submitted version.
FUNDING
All the procedures were performed as part of the employment
of the authors OZ, EG, LP, VR, DL, SLB, and SB at San Michele
Veterinary Hospital, GA in Besta Institute, FP at European
Institute of Oncology, and RP, MD, VC, and AP at Milan
University, CT in Lipogems International.
ACKNOWLEDGMENTS
MD was supported by the Ph.D. program in Molecular and
Translational Medicine, Università degli Studi di Milano,
Italy. The authors are grateful to Dr. Paolo Montuschi
for the illustration of Figure 1 and thank the Laboratory
of Histopathology, Istituto Zooprofilattico Sperimentale
delle Venezie (PD-Italy), for the information regarding
immunohistochemistry protocol.
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Conflict of Interest: CT is the scientific director of Lipogems International.
The remaining authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a potential
conflict of interest.
Copyright © 2021 Zeira, Ghezzi, Pettinari, Re, Lupi, Benali, Borgonovo, Alessandri,
Petrella, Paroni, Dei Cas, Tremolada, Coccè and Pessina. This is an open-access
article distributed under the terms of the Creative Commons Attribution License (CC
BY). The use, distribution or reproduction in other forums is permitted, provided
the original author(s) and the copyright owner(s) are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice.
No use, distribution or reproduction is permitted which does not comply with these
terms.
Frontiers in Veterinary Science | www.frontiersin.org 7January 2021 | Volume 7 | Article 585427
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