Intratumoral Cancer Chemotherapy with a Carrier-Based Immunogenic Cell-Death Eliciting Platinum (IV) Agent

Article abstract of DOI:10.1021/acs.molpharmaceut.0c00781

A carrier-based, immunogenic cell death (ICD)-eliciting platinum(IV) chemotherapeutic agent was synthesized via complexation between an axially derivatized Pt(IV)-tocopherol and hyaluronan (HA)-tocopherol nanocarrier. The resultant HA-Pt(IV) complex demonstrated antiproliferative activity and induced calreticulin translocation, an indicator of ICD, in murine and human head and neck cancer (HNC) cells. The intratumorally administered HA-Pt(IV) treatments were tolerable and efficacious in both immunocompetent and immunodeficient mice with HNC, partially because of the direct cytotoxicity. Superior efficacy and survival were observed in the immunocompetent group, suggesting a possible Pt(IV)-induced immunological response, which would only manifest in animals with an intact immune system. Subsequent imaging of tumor tissues demonstrated increased macrophage infiltration in the HA-Pt(IV)-treated tumors compared to the nontreated controls and the cisplatin-treated tumors, suggesting favorable inflammatory activation. RNA sequencing of HA-Pt(IV)-treated tumors indicated that carbohydrate and vitamin metabolisms were the most important Kyoto Encyclopedia of Genes and Genomes pathways, and molecular function, biological process, and cellular component were highly enriched gene ontology categories.

Full text of DOI:10.1021/acs.molpharmaceut.0c00781

Subscriber access provided by CORNELL UNIVERSITY LIBRARY
Article
Intratumoral cancer chemotherapy with a carrier-based
immunogenic cell death eliciting platinum (IV) agent
Chad Groer, Ti Zhang, Ruolin Lu, Shuang Cai, Derek Mull, Aric Huang,
Melanie Forrest, Cory J. Berkland, Daniel Aires, and Marcus Laird Forrest
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.0c00781 • Publication Date (Web): 25 Sep 2020
Downloaded from pubs.acs.org on September 26, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 25
Molecular Pharmaceutics
1
2
3
O
O
O
4
5
6
7
8
9
O
O
O
H3N
H3N
Cl
Cl
Pt
NH
OH
O
O
NH
OH
O
O
O
O
O
HO
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
OH
NH
Structure of HA-Pt4
O
n
HA-Pt4 and oxaliplatin elicit immunogenic cell death,
illustrated by calreticulin translocation (green fluorescence).
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 2 of 25
1
2
3
4
5
Intratumoral cancer chemotherapy with a carrier-based immunogenic cell death
eliciting platinum (IV) agent
6
7
8
9
Chad Groer1, Ti Zhang1, Ruolin Lu2, Shuang Cai1, Derek Mull1, Aric Huang2, Melanie Forrest1,
Cory Berkland2,3, Daniel Aires1,4, Marcus Laird Forrest1,2*
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1HylaPharm LLC, 2029 Becker Dr, Lawrence KS 66047, USA
2Department of Pharmaceutical Chemistry, The University of Kansas, 2095 Constant Ave,
Lawrence, Kansas 66047, USA
3Department of Chemical and Petroleum Engineering, The University of Kansas, 1530 W
15th St, Lawrence, Kansas 66045, USA
4Division of Dermatology, Department of Internal Medicine, School of Medicine, The
University of Kansas Medical Center, 3901 Rainbow Blvd, Kansas City, Kansas 66160, USA
*Corresponding Author:
Dr. Marcus Laird Forrest
lforrest@ku.edu
Tel: 785-864-4388
Fax: 785-289-8131
Abstract
A carrier-based, immunogenic cell death (ICD)-eliciting platinum(IV) chemotherapeutic
agent was synthesized via complexation between an axially derivatized Pt(IV)-tocopherol
and hyaluronan-tocopherol nanocarrier. The resultant HA-Pt(IV) complex demonstrated
anti-proliferative activity and induced calreticulin translocation, an indicator of ICD, in
murine and human head and neck cancer cells. The intratumorally administered HA-Pt(IV)
treatments were tolerable and efficacious in both immunocompetent and immunodeficient
mice with head and neck cancer, partially due to the direct cytotoxicity. Superior efficacy and
survival was observed in the immunocompetent group, suggesting a possible Pt(IV)-induced
immunological response, which would only manifest in animals with an intact immune
system. Subsequent imaging of tumor tissues demonstrated increased macrophage
infiltration in the HA-Pt(IV) treated tumors compared to the non-treated controls and the
cisplatin-treated tumors, suggesting favorable inflammatory activation. RNA sequencing of
HA-Pt(IV)-treated tumors indicated that carbohydrate and vitamin metabolisms were the
most important Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and molecular
function, biological process and cellular component were highly enriched gene ontology
(GO) categories.
Keywords
Head and neck cancer; platinum (IV) chemotherapy; hyaluronic acid; immunogenic cell
death; RNA sequencing, KEGG and GO analysis
ACS Paragon Plus Environment

Page 3 of 25
Molecular Pharmaceutics
1
2
3
4
Introduction
Head and neck cancers (HNCs) are the sixth most common cancer in people worldwide with
over 630,000 new diagnoses each year and with an estimated incidence rate of 65,000 in the
United States alone . HNCs are categorized based on the anatomical sites where they
5
6
7
8
1
originate, such as the oral cavity (lips, tongue, gums, hard palate, floor of the mouth), nasal
2
9
cavity and paranasal sinuses, salivary glands, pharynx (throat), and larynx (vocal cords) .
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Treatment options include conventional modalities of surgery, radiation therapy and
chemotherapy. Newer targeted therapies such as anti-epidermal growth factor receptor
(EGFR) antibodies (e.g. Cetuximab) inhibit the stimulation of EGFRs in SCC, and checkpoint
inhibitor immunotherapies disrupt the interactions between the programmed cell death
protein 1 (PD1) and the programmed death ligand 1 (PDL1) (e.g. Pembrolizumab and
Nivolumab) 3. The selection of treatment modalities depends primarily on the location and
the stage of the HNCs, in which concurrent and sequential uses of multiple modalities are
common practices in the paradigm of HNC oncology.
In this report, the authors discuss the development and application of an intralesional
(intratumoral) platinum(IV) chemotherapeutic for HNCs via direct tumor injection. The
current work is based on previous preclinical and canine clinical studies of an intralesional
platinum(II) delivery system, in which hyaluronan-conjugated cisplatin (HylaPlat™) was
administered intratumorally in laboratory mice with HNCs and pet dogs with spontaneous
cancers including HNCs. The intralesional HylaPlat™ demonstrated sustained release and
prolonged retention of platinum in the lesion and the surrounding lymphatic basin in mice
and rats 4-9, and a remarkably high ratio of platinum in the tumor tissues compared to the
systemic circulation in dogs 10. The improved targeted distribution of HylaPlat™ translated
into anti-cancer efficacy against HNCs in laboratory mice 11-13 and in pet dogs with naturally
occurring malignancies 14, 15. HylaPlat™ did not induce histologically evident injection-site
reactions and systemic side effects such as renal and hepatic toxicities in rodents. Canine
clinical studies, however, revealed myelosuppression, neutropenia, and liver injury, possibly
due to the reactivity of the liberated platinum(II) from the loosely bound hyaluronan carriers
in the form of diaquated cisplatin that subsequently entered circulatory system, bone
marrow, and liver 14. To improve the toxicology and the pharmacodynamics of intralesional
platinum(II) therapy, an effective and safer platinum(IV) version of HylaPlat™ was
developed.
Platinum(IV) chemistry has been sought by medicinal chemists for several decades and
various mono-, di- and mixed axial-functionalized molecules have been synthesized as
platinum(II) prodrugs 16-19. The octahedrally coordinated platinum(IV) is more inert
chemically than the planar platinum(II), making it more resistant to degradation and
interactions with off-target biological substances in blood circulation and normal tissues
before reaching the tumor site. However, partitioning and accumulation of drug in red blood
cells, and subsequent hemoglobin Fe reduction to Pt(II) inactivates drugs such as Satraplatin
(JM216) 20, which hinders their clinical use. To circumvent this shortcoming, we hypothesize
that local, intralesional delivery of platinum(IV) directly to the tumor tissue may be
advantageous. Once administered intratumorally, Pt(IV) accesses cancer cells that have a
more reducing environment (e.g. in cytoplasm) and higher concentrations of reductants like
ascorbic acid and glutathione, to which it binds, and is reduced to the active, DNA-damaging
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 4 of 25
1
2
3
4
5
6
7
8
9
Pt(II) moieties 19, 21. Furthermore, the reduction potentials and reduction rates of Pt(IV)
compounds could be tailored by installing different axial groups to the metal center. For
example, carboxylato ligands display intermediate reduction potentials, whereas hydroxido
ligands exhibit low and chlorido ligands exhibit high reduction potentials, which, in
consequence, yield Pt(IV) molecules with different reactivity in vivo 20, 22
.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In addition to improved kinetic stability, Pt(IV) may differ from Pt(II) in its mechanism of
action. Traditionally, it is well recognized that platinum agents form Pt-DNA adducts and
cause inhibition in protein biosynthesis which lead to apoptotic death of cancer cells.
Moreover, Pt(IV) treatment was also shown to induce immunogenic cell death (ICD) and
trigger massive infiltration of cytotoxic CD8+ T lymphocytes in colon cancer 23. In contrast to
a non-immunogenic cell death, ICD activates the host’s immune system against the invading
cancer. The dying cancer cells undergo several immunogenic processes, including
calreticulin translocation to the cell surface, release of ATP and HMGB1, and generation of
onco-antigens, that collectively result in the maturation of dendritic cells and the activation
of T lymphocytes 24, 25. Similar findings were reported for oxaliplatin 24, 26, a Pt(II) agent with
a bidentate ligand that shares structure similarity with Pt(IV) molecules. Accordingly, we
hypothesize that a carrier-based Pt(IV) complex could elicit immunogenic cell death in a
similar manner once the Pt(IV) cargo enters the cancer cells, resides and activates in the
reductive cytoplasm.
Materials and Methods
Cis-Dichlorodiamine platinum (II) (cisplatin) was purchased from Strem Chemicals, Inc.
(Newburyport, MA). Sodium hyaluronate was purchased from Contipro Inc (Dolní Dobrouč,
Czech Republic). 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-
oxide hexafluorophosphate (HATU), and 3-(3-Dimethylaminopropyl)-1-ethyl-carbodiimide
hydrochloride (EDC-HCl) was purchased from Chem-Impex International, Inc. (Wood Dale,
IL). 1-Hydroxybenzotriazole hydrate (HOBtH2O) was purchased from Advanced ChemTech
(Louisville, KY). N-(tert-Butoxycarbonyl)glycine (Boc-Gly-OH), hydrogen peroxide solution
(H2O2,
30
wt.%
in
H2O),
succinic
anhydride,
(±)-α-tocopherol,
N,N′-
Dicyclohexylcarbodiimide (DCC), N,N-Dimethylpyridin-4-amine (DMAP), trifluoroacetic acid
(TFA), N,N-Diisopropylethylamine (DIPEA), oxaliplatin, tetrabutylammonium hydroxide
solution (TBA-OH, 40 wt.% in H2O), and anhydrous organic solvents were purchased from
Sigma-Aldrich (Saint Louis, MO). Alexa Fluor ® 488 anti-CD8a, Alexa Fluor® 594 anti-
CD11b, and Alexa Fluor® 647 anti-CD11c antibodies were purchased from BioLegend (San
Diego, CA). All other chemicals and organic solvents were purchased from Thermo Fisher
Scientific (Waltham, MA).
1. Synthesis of Pt(IV)-Tocopherol [Pt(IV)-Toco] conjugate, (4)
The synthetic scheme is shown in Figure 1. A bi-conjugated tocopherol Pt(IV) was reported
by Suntharalingam et al 27, in which doubly substituted Pt(IV)(toco)2 and Pt(IV)(toco)(OEt)
were synthesized. Different from the reported molecules, we found that an axial substitution
with a single tocopherol and a simple hydroxyl placement at the opposite axial position
renders the characteristics needed for further fabrication of the carrier-based prodrug
without the additional lipophilicity and bulk.
ACS Paragon Plus Environment

Page 5 of 25
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Synthetic scheme of Pt(IV)-Tocopherol [Pt(IV)-Toco] conjugate. a, water, H2O2, 50
°C; b, DMSO, 25 °C; c, Boc-Gly-OH, DCC, DMAP, DCM; d, TFA, DCM; and e, HATU, DIPEA, DMSO.
1.1 Synthesis of cis,cis,trans-[Pt(NH3)2Cl2(OH)2], (1)
Fifty milliliter of H2O2 (30 wt% in water) was added dropwise to a stirred suspension of
cisplatin (1.05 mg) in 3 mL of water (50 °C) in the dark. After 1 hour, a light-yellow
suspension was obtained and subjected to recrystallization at 4 °C overnight. The product
was collected by filtration, sequentially washed with ice-cold water, ethanol and diethyl
ether, and dried under vacuum. A bright yellow solid was obtained as a product (95% yield).
1.2 Synthesis of cis,cis,trans-[Pt(NH3)2Cl2(OOCCH2CH2COOH(OH)] (Pt(IV)-Su, 2)
A suspension of compound 1 (200 mg, 0.60 mmol) and succinic anhydride (66 mg, 0.66
mmol) in 16-mL of anhydrous DMSO was stirred in the dark at 25 °C overnight and resulted
in a clear light yellowish solution. After DMSO was removed by lyophilization (Labconco 2.5
Plus FreeZone, Kansas City, MO), the product was obtained by recrystallization from acetone
at -20 °C overnight, and washed with cold acetone and diethyl ether. The solid was dried
under vacuum and was a pale yellow product (70%). 1H NMR (400 MHz, DMSO-d6) δ 6.24–
5.66 (m, 6H), 2.34–2.45 (m, 4H), (Figure S1).
1.3 Synthesis of H-Gly-Tocopherol (H-Gly-Toco, 3)
Boc-Gly-OH (0.89 g, 5.11 mmol), (±)-α-tocopherol (2.42 g, 5.62 mmol), and DMAP (64 mg,
0.52 mmol) were dissolved in 20-mL of anhydrous DCM. The mixture was added dropwise
to a solution of DCC (1.16 g, 5.62 mmol) in 5-mL of anhydrous DCM, and was stirred at room
temperature overnight. The reaction mixture was then stored at -20 °C for 2 hours. After the
white precipitate was filtered off, solvent was removed under vacuum to obtain a pale-
yellow syrup. The product was purified by a Combiflash Rf+ purification system (Teledyne
ISCO, Lincoln, NE), and eluted at 5% v/v of EtOAc in hexane. The product fractions were
concentrated under vacuum to obtain a colorless syrup (92% yield).
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 6 of 25
1
2
3
4
5
6
7
8
9
1HNMR (400 MHz, Chloroform-d) δ 5.17 (s, 1H), 4.21 (d, J = 5.0 Hz, 2H), 2.59 (t, J = 6.8 Hz,
2H), 2.10 (s, 3H), 2.02 (s, 3H), 1.98 (s, 3H), 1.79 (ddq, J = 20.0, 13.3, 6.8 Hz, 2H), 1.63–1.49
(m, 3H), 1.49–1.45 (m, 9H), 1.38 (qd, J = 8.4, 7.5, 3.0 Hz, 4H), 1.31–1.18 (m, 11H), 1.17–1.02
(m, 6H), 0.87 (d, J = 6.6 Hz, 6H), 0.85–0.83 (m, 6H) (Figure S2); 13C NMR (101 MHz, CDCl3) δ
169.31, 155.83, 149.78, 140.30, 126.71, 125.00, 123.33, 117.64, 80.24, 75.28, 42.47, 39.52,
37.70, 37.61, 37.56, 37.54, 37.44, 32.93, 32.85, 31.17, 28.46, 28.13, 24.96, 24.59, 22.87, 22.77,
21.17, 20.73, 19.90, 19.83, 13.12, 12.27, 11.96. (Figure S3); Chemical formula: C36H61NO5.
ESI-MS: M + Na+ = 610.4447, found 610.4444.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A solution of TFA (5 mL) in DCM (5 mL) was added dropwise into a solution of Boc-Gly-Toco
(2.87g, 4.89 mmol) in DCM (15 mL) at 0 °C for 30 min, and the reaction mixture was stirred
at ambient temperature overnight. The reaction mixture was evaporated under vacuum, and
the resulting gray solid was purified over silica. The product (H-Gly-Toco, 3) fractions
1
yielded a white solid after concentration under vacuum (yield 60%). H NMR (400 MHz,
DMSO-d6) δ 8.45 (s, 2H), 4.27 (s, 2H), 2.57 (t, J = 6.9 Hz, 2H), 2.04 (s, 3H), 1.97 (s, 3H), 1.95
(s, 3H), 1.81–1.72 (m, 2H), 1.56–1.44 (m, 3H), 1.40 (t, J = 8.7 Hz, 4H), 1.31–1.16 (m,11H),
1.16–0.99 (m, 6H), 0.83 (dd, J = 9.1, 6.5 Hz, 12H), (Figure S4); 13C NMR (101 MHz, DMSO) δ
166.76, 149.01, 139.57, 126.21, 124.91, 122.07, 117.59, 74.95, 36.73, 36.58, 32.04, 31.93,
27.37, 24.13, 23.69, 22.54, 22.45, 20.31, 19.87, 19.61, 19.55, 12.77, 11.91, 11.56, (Figure S5).
Chemical formula: C31H53NO3. ESI-MS: M+H+ = 487.4025, found 487.4106.
1.4 Synthesis of Pt(IV)-Su-Gly-Toco (Pt(IV)-Toco, 4)
A mixture of Pt(IV)-Su (0.54 g, 1.25 mmol), HATU (0.71 g, 1.87 mmol), and DIPEA (0.43 ml,
2.47 mmol) in 30 mL of anhydrous DMSO was added to a solution of H-Gly-Toco (0.62 g, 1.28
mmol) in DMSO and stirred overnight. The DMSO was removed under reduced pressure, and
the resulting oily mixture was purified by the Combiflash Rf+ system eluting at 0% to 20%
of MeOH in DCM. The product fractions were concentrated under vacuum to a brown solid,
and subsequently washed with MeOH to obtain the product as a beige solid (25%). 1H NMR
(400 MHz, DMSO-d6) δ 8.47 (dt, J = 16.9, 5.9 Hz, 1H), 6.17–5.62 (m, 6H), 4.14 (t, J = 5.0 Hz,
2H), 2.63–2.52 (m, 3H), 2.48–2.35 (m, 4H), 2.02 (s, 3H), 1.93 (s, 3H), 1.91 (s, 3H), 1.76 (t, J =
7.7 Hz, 2H), 1.51 (dt, J = 13.1, 6.5 Hz, 3H), 1.44–1.34 (m, 4H), 1.32–1.17 (m, 11H), 1.17–1.02
(m, 6H), 0.92–0.80 (m, 12H), (Figure S6).
2 Synthesis of Hyaluronan-Tocopherol (HA-Toco) conjugate
ACS Paragon Plus Environment

Page 7 of 25
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Synthetic scheme of HA-Toco.
2.1 Synthesis of HA-TBA, (5)
Sodium hyaluronate (32 kDa, 10g) was dissolved in 200 mL of water, and stirred for 1 hour.
The solution was added to 50 g of AG 50W-X8 cation-exchange resin (20-50 mesh, H-form,
Bio-Rad, Hercules, CA), and stirred overnight. The mixture was filtered, and the filtrate was
titrated with TBA-OH to pH 8-9. The aqueous solution was lyophilized to obtain a white
sponge-like cake and was stored at -20 °C for future use (Yield 64%).
2.2 Synthesis and characterization of HA-Toco, (6)
A solution of HA-TBA (200 mg, 0.32 mmol) in 12 mL of DMF was stirred for 30 min at ambient
temperature (ca. 20°C). A mixture of H-Gly-Toco (15.6 mg, 0.032 mmol), EDC (15.4 mg, 0.08
mmol), and HOBtH2O (7.4 mg, 0.048 mmol) in 2.3 mL of DMF was added to the previous
solution at ambient temperature and stirred overnight. The reaction mixture was dialyzed
using regenerated cellulose dialysis tubing (10,000 MWCO, Thermo scientific) against 50
vol.% EtOH in water overnight, then against NaCl dialysate (150 mM, 2 changes in 24 hours),
and subsequently against water (3 changes in 2 days). The dialyzed mixture was lyophilized
to yield a white cotton-like polymer 6 (117 mg, yield 69%). The degree of substitution (SD)
of tocopherol molecules on HA was calculated to be ca. 6% on a molar basis by comparing
the peak integration ratio of the N-acetyl group of HA (s, 3H, 2.02 ppm) to the methyl groups
1
of tocopherol (m, 12H, 0.81-0.86 ppm) in HNMR spectra (Figure S7). The hydrodynamic
diameter of HA-Toco in an aqueous solution was determined using a ZetaPALS (Brookhaven
Instrument Corporation, Holtsville, NY). All measurements were carried out with five
replicates.
3 Preparation of HA-Toco/Pt(IV)-Toco complex
HA-Toco (122 mg) was rehydrated in 12 mL of water on a shaker overnight. To the HA-Toco
solution, a solution of Pt-Toco (61 mg) in 2.4 mL of DMSO was added dropwise, and the
resulting mixture was stirred for 2 hours in the dark. The solution was lyophilized overnight
to remove water and DMSO, then the solid was rehydrated with 5 mL of water for injection
(WFI) into a homogenous nanosuspension. The Pt concentration was determined by an
inductively coupled plasma-mass spectrometry (ICP-MS, Agilent Technologies 7500i, Santa
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 8 of 25
1
2
3
4
5
6
7
8
Clara, CA) using terbium as the internal standard 28. High purity argon (>99.996%) was used
as the carrier gas. The final drug concentration was calculated on a cisplatin weight basis.
The loading degree of Pt on HA-Toco/Pt(IV)-Toco is approximately 10%. Note: for simplicity,
the HA-Toco/Pt(IV)-Toco is abbreviated as HA-Pt(IV) in this article.
9
4 In vitro studies
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4.1 AT84 cells
AT84 cells were derived from a spontaneous squamous cell carcinoma in the oral mucosa of
29, 30
a C3H mouse
and were gifted by Aldo Venuti (Regina Elena National Cancer Institute,
Rome, Italy). Cells tested negative for interspecies contamination (Idexx BioResearch) and
rodent pathogens (21 pathogen IMPACT I PCR profile), and negative for Mycoplasm
contamination prior to animal studies (Lonza, MycoAlert test kit). Idexx CellCheck STR
(short tandem repeat) profile: MCA-4-2: 20.3, 21.3; MCA-5-5: 15; MCA-6-4: 18, 19; MCA-6-7:
12; MCA-9-2: 15; MCA-12-1: 16; MCA-15-3: 25.3, 26.3; MCA-18-3: 16; MCA-X-1: 26, 27. Cells
were cultured in RPMI-1640 media (Gibco, Thermo Fisher Scientific, Waltham, MA)
supplemented with 10% FBS (Corning Corning, NY), 100 U/mL penicillin, and 100 µg/mL
streptomycin (HyClone, Thermo Fisher Scientific, Waltham, MA) in a humidified incubator
at 37°C and 5% CO2.
4.2 Anti-proliferation
AT84 cells were seeded into 96-well plates (3,000 cells/well) and allowed to attach
overnight. Cisplatin and HA-Pt(IV) were diluted in water and added to the cells (8
concentrations in duplicate). After 72 hours at 37oC, Resazurin Blue (Acros Organics, Geel,
Belgium) in PBS was added to each well (5 µM final concentration) and incubated at 37oC for
4 hours. Fluorescence (λex 550 nm, λem 605 nm) was quantified with a SpectraMax Gemini
XS plate-reader (Molecular Devices, Sunnyvale, CA). The relative growth of cells incubated
with each compound concentration was normalized to vehicle-treated controls (100%
proliferation). Data from at least three separate experiments were analyzed using non-linear
regression (GraphPad Prism 5.0) to generate IC50 values.
4.3 Calreticulin translocation
Plasmid: A mouse calreticulin-HaloTag®-KDEL fusion protein (reported by Golden et al. 31)
was synthesized (ATUM, Newark, CA) and inserted into the pD643 mammalian expression
vector having a CayenneRFP fluorescent reporter gene (Ex:554nm / Em:590nm) (Addgene,
Watertown, MA, USA, plasmid #153529). The resulting construct was sequenced to confirm
the insert.
AT84 cells were plated in 6-well culture plates and allowed to grow to ~90% confluence.
Cells were transfected with the calreticulin plasmid with lipofectamine (Invitrogen,
Carlsbad, CA) using 10 µg/well pDNA. After 5 hours at 37°C, cells were split 1:3 into 6-well
plates with complete media and incubated overnight at 37°C. Cisplatin, oxaliplatin, or HA-
Pt(IV) was added to a final concentration of 500 µM (drug). Following 12-15 hours
incubation, cells were washed with fresh media. Cells were incubated at 37°C for 30 minutes
with 1 µM HaloTag® Alexa Fluor® 488 Ligand (1 mM solution, Promega, Madison, WI) and
20 µM Hoechst dye (20 mM solution, Thermo Scientific, Rockford, IL), washed with media,
ACS Paragon Plus Environment

Page 9 of 25
Molecular Pharmaceutics
1
2
3
4
5
6
and imaged within one hour. Live cells were imaged using an Olympus IX81 inverted
epifluorescence microscope with a 60x objective. Representative images are shown from 3
independent experiments (Figure 4).
7
8
9
5 In vivo studies
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5.1 Mice
All rodent studies were performed at the University of Kansas Animal Care Unit, which is in
compliance with the “Guide for the Care and Use of Laboratory Animals” and is accredited by
the Association for the Assessment and Accreditation of Laboratory Animal Care
International (AAALAC). The studies were done according to a protocol approved by the
University of Kansas IACUC committee.
5.2 Immuno-competent tumor model for efficacy
Wildtype male and female C3H mice (Charles River Strain 025, 6-8 weeks old, 20-25g) were
used for in vivo tumor studies. Since no differences were found between the sexes, the
reported results combined both sexes into one group. Mice were anesthetized using 5%
isoflurane in O2 for 5 minutes. One million AT84 cells in 50 µl PBS were injected
subcutaneous (s.c.) into the floor of the mouth via an extra-oral route of C3H mice to obtain
orthotopic allograft tumors 29, 30. Treatment began when tumors reached 75 mm3, generally
on days 4 to 7 after cell injection. Under isoflurane anesthesia, HA-Pt(IV) (3 mg/kg Pt(IV)
basis) or vehicle (50 µl hyaluronan-tocopherol, 16.7 mg/mL) was injected intratumoral once
per week for 3 weeks. Tumor size was calculated: tumor volume (mm3) = 4π/3 × (width/2)2
× (length/2) 32, where length is the longer of two perpendicular dimensions. Statistical
comparisons were conducted using GraphPad Prism v.7 software (San Diego, CA).
5.3 Immuno-deficient tumor model for efficacy
Nude male and female mice (Charles River Ath/nu, 6-8 weeks old, 20-25g) were used for in
vivo xenograft studies. Since no differences were found between the sexes, results show
both sexes as one group. Mice were anesthetized using 5% isoflurane in O2 for 5
minutes. One million MDA-1986 cells in 50 µl PBS were injected subcutaneously into the left
cheek of mice to obtain orthotopic xenograft tumors 11, 13. Treatment began when tumors
reached 75 mm3, generally on day 14 after cell injections. Under isoflurane anesthesia, HA-
Pt(IV) (3 mg/kg Pt(IV) basis) or vehicle (1.5 MDa hyaluronan, 16.7 mg/mL) was injected
intratumoral once per week for 3 weeks. Tumor size and statistical comparisons were
performed as stated in the C3H model.
5.4 Tumor stains using immune cell markers
Mice with AT84 derived head and neck tumors were randomly divided into 5 groups,
including 1) no treatment, 2) three weekly intravenous cisplatin injection (3 mg/kg per
dose), 3) one intratumoral HA-Pt(IV) injection (3 mg/kg), 4) two weekly intratumoral HA-
Pt(IV) injections (3 mg/kg per dose), and 5) three weekly intratumoral HA-Pt(IV) injections
(3 mg/kg per dose). Our previous studies showed that intravenous cisplatin penetrated
tumors inadequately and cleared rapidly from the systemic circulation, so IV cisplatin was
administered as a triple-dose treatment instead of a double- or single-dose treatment. All
mice, except for the no treatment group, were sacrificed after the last injection. The
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 10 of 25
1
2
3
4
5
6
7
8
9
individual tumors were excised and frozen in OCT medium. The embedded tumors were
sectioned into 20 µm slices on a Shandon Cryotome® FSE (Thermo Scientific). The sections
were fixed in 10% formalin for 15 minutes, blocked with 5% goat serum in PBS, and stained
with primary antibodies. Fluorescent-dye conjugated primary antibodies were diluted to 5
µg/mL in blocking buffer (5% goat serum in PBS) and incubated overnight at 4 °C. After
antibody staining, sections were stained with 10 μg/mL Hoechst 33342 for 15 minutes,
mounted in SouthernBiotech™ Fluoromount-G™ Slide Mounting Medium, and stored in the
dark at 4 °C. Images were acquired using an Olympus IX-81 inverted epifluorescence
microscope at 10x magnification. The acquired images were compiled on Slidebook 6.0.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5.5 RNA isolation
Tumor pieces were stored in RNAlater solution at room temperature for at least one day and
then at 4°C until RNA isolation. Tumor pieces were removed from RNAlater solution and
homogenized in 10 mL TRIzol reagent (38% phenol, 0.8 M guanidine thiocyanate, 0.4 M
ammonium thiocyanate, 0.1 M sodium acetate, 5% glycerol). Homogenate was divided into
1 mL aliquots for ease of subsequent centrifugation steps. To each 1 mL of homogenate, 100
µl BCP (Molecular Research Center, Cincinnati, OH) was added, shaken vigorously 15
seconds and centrifuged 12,000 x g, 10 minutes, 4°C. Top aqueous layer contains RNA, which
is precipitated with isopropanol and washed with 75% ethanol. RNA was reconstituted in 1
mM sodium citrate pH6.4 and stored at -80°C. RNA quality was analyzed using the Agilent
TapeStation; all RNA samples showed an RNA integrity number greater than 8, which is
indicative of excellent quality.
5.6 RNA sequencing
Sequencing was performed on an Illumina NextSeq 550 in the SR-75 mode (KU Genome
Sequencing Core). Three reads from each demultiplexed sample were combined and checked
for quality in FastQC v0.11.8.33 The expression of the transcripts was quantified using
Salmon v.1.0.0 with an index compiled from the Mus musculus GRCm38 primary assembly
34, 35
and transcriptome.
The transcript abundance estimates from Salmon was converted
into gene-level count matrices using tximport v.1.14.2.36 Count normalization was
performed using DESeq2 v.1.26.0., and data was filtered by p-value < 0.1 and log2 fold change
(LFC) > 1.58 to obtain genes with most significant differences in expression between control
and Pt4-treated tumors. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes
(KEGG) pathways clustering analysis was performed on the expression data using Gene Set
Enrichment Analysis (GSEA) v 3.0.0 37, 38. Sequence data and sample descriptions are
available at NCBI BioProject accession #PRJNA612618.
Results and Discussion
The carrier-based HA-Pt(IV) was developed first by functionalizing one of the axial ligands
of the platinum with activated tocopherol via a succinic linkage. Subsequently, the
derivatized lipophilic Pt(IV) molecule was complexed with a hyaluronan-tocopherol
conjugate via attractions between the lipophilic tocopherol portions of the two molecules,
forming a hyaluronic acid polymer-based delivery platform for the anticancer Pt(IV) cargo.
A Pt(IV)-based oncology molecule is of interest in this study due to its less toxic nature than
ACS Paragon Plus Environment

Page 11 of 25
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
the platinum(II) compounds as a result of the altered reduction kinetics of the platinum(IV)
39, making them less likely to cause dose-limiting side effects as seen in cisplatin, as well as
due to its improved cellular accumulation and greater lipophilicity, a property facilitating the
21
encapsulation or complexation into nanocarriers . Lippard et al. at MIT reported several
derivatives of Pt(IV) that utilized tocopherol or fatty acids to encapsulate the drugs in
micelles or form other prodrugs 27, 40. Pt(IV)-containing nanoconjugates such as dextran-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
platinum(IV) and ethylenediamine modified hyaluronic acid-platinum(IV) were also
41, 42
reported by other groups and underwent preclinical development
.
Based on our experience with carrier-based Pt(II) drugs, we hypothesized that localized
delivery to tumor would further improve the activity of Pt(IV). In addition, we believed a
hyaluronan-based carrier could increase retention and tumor-associated lymph node
uptake, while lessening RBC inactivation, a drawback of previous unsuccessful Pt(IV)
molecules including the clinically investigated drug Sartaplatin.43, 44 The uniqueness of the
HA-Pt(IV) formulation in this work is manifested by the combination of at least three factors
including - a lipophilic platinum(IV) and its immunological properties as discussed below, a
safe and pharmacokinetically tailored hyaluronan-based drug carrier demonstrated by
previous animal studies 7-9, 11, 13, 45-47, and a clinically feasible mode of intralesional delivery
for spontaneous cancers based on the results of canine clinical trials 10, 14, 15. The HA-Pt(IV)
formulation uses FDA-designated generally recognized as safe (GRAS) substances such as α-
tocopherol, and biodegradable and biocompatible natural polysaccharide such as hyaluronic
acid, which has been approved by the FDA for uses as a dermal filler and as an intra-articular
injection. These characteristics render HA-Pt(IV) a clinically relevant molecule and justify
the synthetic and testing efforts, which are discussed below in detail.
1 Hydrodynamic diameter of HA-Toco
The hydrodynamic diameter of HA-Toco in water solution was determined to be 332.0 ± 2.2
nm with a polydispersity of 0.145 ± 0.028 using the intensity weighted Gaussian distribution.
The particle size of tocopherol functionalized hyaluronan nanoparticles was much larger
than the non-tocopherol conjugated hyaluronic acid molecules previously evaluated by the
48
authors (25.2 ± 4.4 nm) . The formation of larger particles can be attributed to the intra-
and inter-molecular interactions between the hydrophobic tocopherol pedants, tangling the
long and linear HA chains. These HA-toco nanoparticles further attract small-molecule
Pt(IV)-tocopherol and eventually construct drug-retaining depots that can release Pt(IV) and
cause growth inhibition of cancer cells.
2 In vitro studies
2.1 Anti-proliferation
To determine whether HA-Pt(IV) exhibits anti-proliferative activity against cancer cells, it
was tested for growth inhibition of the AT84 murine head and neck cancer cell line (Figure
3) and the human MDA-1986 head and neck cancer cells. The positive control, cisplatin, and
HA-Pt(IV) both inhibited over 80% of cell growth compared to vehicle-treated controls at
the highest doses tested with potencies (IC50) of 3.0 ± 0.26 µM and 6.1 ± 1.1 µM, respectively.
Oxaliplatin also inhibits growth of these cells with slightly lower potency (13.6 ± 3.8 µM).
The results suggest that the HA-Pt(IV) complexes demonstrated similar growth inhibition
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 12 of 25
1
2
3
4
5
against cancer cells compared to cisplatin, enabling further mechanistic evaluation such as
the calreticulin translocation assay discussed below.
6
7
8
9
100
80
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
60
40
HA-Pt(IV)
20
cisplatin
oxaliplatin
0
-8
-7
-6
-5
-4
-3
Log [M]
Figure 3. HA-Pt(IV) inhibits growth of head and neck cancer cells. AT84 cells were cultured
in the presence of increasing concentrations of compounds for 72 hours and cell
proliferation was quantified. Data from at least five separate experiments was analyzed by
non-linear regression. Representative dose response curves are shown. All drugs show full
efficacy. Potencies are HA-Pt(IV) IC50 = 6.1 ± 1.1 µM, cisplatin IC50 = 3.0 ± 0.3 µM, and
oxaliplatin IC50 = 13.6 ± 3.8 µM (n=7-8).
2.2 Calreticulin translocation
The immunological effects induced by certain anticancer chemotherapeutics began to divert
the interest of developers of conventional chemotherapies due to their pathways for the
activation of the immune system against the cancer. Chemotherapy can induce cell death that
can be either immunological or non-immunological. Cisplatin causes non-immunogenic cell
death, wherein apoptotic cell debris is cleared by macrophages without the activation of
dendritic cells or the release of significant levels of pro-inflammatory cytokines such as IL12
and TNFα 49, while other chemotherapies, such as oxaliplatin, cause immunologic cell death
24, 25, 50, 51. ICD is a particular type of apoptotic cancer cell death that activates the host
24, 52
immune system
against tumor cells. Some chemotherapeutics elicit ICD by creating
endoplasmic reticulum stress, leading to membrane translocation of calreticulin (CRT), an
early indicator of ICD, which can be tested in vitro 31, 52. Other characteristics of ICD include
release of ATP and high mobility group protein 1, which activates dendritic cells to present
tumor antigens to the cytotoxic CD8+ T lymphocytes to complete the final destruction of
tumor cells 25. Unlike traditional chemotherapeutics, which rely on the penetration of
nonimmunogenic cytotoxins and sustained exposure, immunogenic chemotherapeutics like
oxaliplatin and Pt(IV) may elicit a tumor-specific immune response 23.
In the calreticulin translocation assay, AT84 cells were transfected with a plasmid coding for
mouse CRT-HaloTag®-KDEL fusion protein. Positivity transfected cells were identified by
visualizing the CayenneRFP reporter protein (red), coded for in the same plasmid. Following
ACS Paragon Plus Environment

Page 13 of 25
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
overnight treatment with an ICD-inducing drug, CRT translocates to the plasma membrane,
exposing the HaloTag® protein to the outside of the cell. Extracellularly exposed HaloTag®
protein can bind to the membrane impermeable HaloTag® Ligand Alexa Fluor® 488. After
overnight treatment with the positive control, oxaliplatin (500 µM) 25, membrane
translocation of CRT is observed as green fluorescence (Figure 4). Similar to oxaliplatin,
Pt(IV) (500 µM) also causes CRT translocation to the membrane, indicating ICD. In contrast,
cisplatin does not cause CRT translocation to the membrane, thus unavailable to bind the
488-labled ligand (no green), indicating a lack of ICD, consistent with previous reports 53. In
an oversimplified sense, the immunogenic oxaliplatin and hopefully the newly developed
HA-Pt(IV) could be regarded mechanistically as the equivalent of cisplatin plus radiation
therapy in terms of inducing ICD (cisplatin does not induce ICD, but irradiation does), which
explains their superior potential for long-term success in a realistic tumor model (e.g.
immunocompetent small laboratory animals, such as C3H mice, or large non-laboratory
animals, such as dogs). While in vitro translocation of CRT is an indicator of ICD, analysis of
in vivo tumor response and immune response (cell types, pro-inflammatory cytokines, etc.)
in an intact animal is required to understand ICD mechanisms. We have begun these in vivo
analyses in this report; more detailed analyses of the ICD immune response remain an active
area of investigation in our lab.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. HA-Pt(IV) causes calreticulin translocation to the cell surface. AT84 cells were
transfected with mouse CRT-HaloTag®-KDEL fusion protein. Positively transfected cells are
identified by the CayenneRFP reporter protein expressed from the same plasmid (red, top
panel). After incubation with drug (500uM; 15 hours), membrane translocation of CRT-
HaloTag® is visualized by addition of the membrane impermeable HaloTag® Ligand Alexa
Fluor® 488 (green, bottom panel). The positive control oxaliplatin and HA-Pt(IV) show CRT
translocation, indicating ICD. In contrast, cisplatin does not show CRT translocation,
consistent with a lack of ICD induction. Live cells were imaged using a 60x objective.
Representative images are shown from 3 independent experiments.
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 14 of 25
1
2
3
4
5
3 In vivo studies
3.1 Mouse tumor efficacy
We used two models of head and neck cancer to determine whether HA-Pt(IV) can inhibit
tumor growth in vivo. One model uses a spontaneously arising oral squamous cell cancer
(AT84) in immunocompetent C3H mice, while the other model uses human derived cervical
nodal metastasis of tongue cancer (MDA1986) in immunodeficient athymic nude mice. C3H
mice with AT84 allografts and nude mice with MDA1986 xenografts were administered HA-
Pt(IV) (3 mg/kg) intratumorally once per week for 3 weeks, starting when the tumor reached
~75 mm3. These doses were empirically determined as tolerable in these mice, resulting in
no decrease in weight or deterioration in body condition.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In both tumor models, HA-Pt(IV) treatments significantly reduced tumor growth over the
time course compared to vehicle treated animals in the control group (Figure 5). In the AT84
allografts, the mean ± SEM for the final tumor measurements of the control and the HA-Pt(IV)
treated were 2027 ± 364 mm3 and 1024 ± 129 mm3, respectively, a 49% tumor inhibition in
an immunocompetent host. On the other hand, the mean ± SEM for the final measurements
of the control and the HA-Pt(IV) treated were 1427±162 mm3 and 812±150 mm3,
respectively, in the MDA-1986 xenografts, a 43% tumor inhibition in an immunodeficient
host.
Though the difference in the percent tumor regression at the end of the study between the
two models was relatively small, the AT84 model showed a difference in tumor growth
between the control and the treated groups much earlier than the MDA1986 model.
Specifically, AT84 tumors in immunocompetent mice were significantly smaller than
controls between day 21 and 32, about one third of the entire study course. In contrast,
MDA1986 tumors in immunodeficient mice were significantly smaller than controls between
days 35 to 42, only 17% of the entire study course. The superior efficacy in the AT84 model
may be due to the ICD effect induced by the Pt(IV) treatment, which would only manifest in
animals with an intact immune system. Pt(IV)-induced ICD may involve dendritic cell
recruitment and T lymphocyte activation to attack and eradicate some of the residual cancer
cells. Thus, the dying cancer cells may act as an immunogenic site to stimulate tumor-specific
immune responses and sustain the effectiveness of the cytotoxic stimulus. Ongoing studies
are examining immune cell responses to HA-Pt(IV) therapy.
ACS Paragon Plus Environment

Page 15 of 25
Molecular Pharmaceutics
1
2
3
4
5
6
AT84, vehicle
AT84, HA-Pt(IV)
MDA1986, vehicle
MDA1986, HA-Pt(IV)
2500
2000
1500
1000
500
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
****
##
****
###
****
*
0
0
7
14
21
28
35
42
Days
Figure 5. HA-Pt(IV) demonstrated growth inhibition of head and neck tumors in
immunocompetent (AT84) and immunodeficient (MDA1986) mouse models (mean ± SEM).
HA-Pt(IV) treatment significantly reduced AT84 tumor growth compared to vehicle controls
in immuno-competent C3H mice (two-way ANOVA, p<0.0001 for treatment, time, and
interaction; Sidak’s multiple comparisons test, *p<0.05,****p<0.0001; n=6-8). In the
immuno-compromised MDA1986 model, HA-Pt(IV) treatment also significantly reduced
tumor growth (two-way ANOVA, p<0.001 for treatment, p<0.0001 time, and p<0.01
interaction; Sidak’s multiple comparisons test, ##p<0.01,###p<0.001; n=8). Individual
growth curves are included in the supplementary data.
3.2 Survival
The HA-Pt(IV) treatment resulted in significantly increased survival in both tumor models
(Figure 6). In the AT84 immunocompetent model, HA-Pt(IV) treatment resulted in a survival
rate of 100% by the time all control mice died. In contrast, in the MDA1986
immunocompromised model, HA-Pt(IV) treatment showed only 50% survival when all
control mice died. The differences observed between the two models’ efficacy and survival
may be partially due to the immunogenic nature of the HA-Pt(IV), though the difference may
also be attributed to the intrinsic characteristics of the two distinct tumor models and
respective growth patterns. The human cancer cell line MDA1986, as opposed to the murine
cancer cell line AT84, was selected to establish the tumor models in nude mice because the
murine cancer cells could grow aggressively in immunodeficient mice and cause premature
casualty, interfering with the validity of the study. Thus, the MDA1986 model was included
for a more thorough examination of the immunological response in tumors.
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 16 of 25
1
2
3
4
5
6
7
8
9
100
80
60
40
20
0
100
80
**
AT84, control
MDA1986, control
AT84, HA-Pt(IV)
MDA1986, HA-Pt(IV)
60
40
20
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
***
0
5
10
15
20
25
30
35
0
14
28
42
56
70
84
Days
Days
Figure 6. Survival rates of MDA-1986 and AT84 tumor models. The control and the HA-
Pt(IV) treated groups differed significantly in both models (Mantel-Cox, **p<0.01,
***p<0.001; n=6-8).
3.3 Tumor immunohistochemistry
Tumor tissues were stained for the immunological markers CD8a, CD11b and CD11c, which
are commonly recognized markers for cytotoxic T lymphocytes, macrophages, and dendritic
cells, respectively. CD8a staining was observed after 2 and 3 weekly injections of HA-Pt(IV)
(2x and 3X), but not after the first injection (1X). A CD8+ cytotoxic T cell response can take
up to 7 days to develop and was not observed 2 days after the first injection, when that group
was sacrificed. For CD11c staining, all groups display a low intensity pattern for dendritic
cells, suggesting a baseline level in all AT84 tumors. In contrast, the CD11b column
demonstrates the most prominent differences between the no treatment and the HA-Pt(IV)
treated groups (1×, 2×, or 3×); to a lesser degree, between the no treatment and the cisplatin
(3×) treated groups. The strongest intensity of fluorescence is detected in the 3× HA-Pt(IV)
treated tumor sections, consistent with this group having the highest accumulated doses of
Pt(IV). In addition, between the 3× HA-Pt(IV) and the 3× cisplatin treated tumors, the former
appears to present a higher number of tumor-infiltrating macrophages than the latter.
Certain chemotherapeutics induce increased macrophage recruitment to tumor
microenvironment 54-56. As a consequence of the inherent plasticity of macrophages, they
respond to local stimulants and allow two contradictory transformations, one resulting in an
inflammatory, M1-polarized activation that inhibits tumor progression, while, the other,
leading to a non-inflammatory, M2-polarized activation that promotes tumor invasiveness
57, 58. In this case, the corresponding local stimulants may either be the trace concentration
of cisplatin, a fraction of its total intravenous dose that manages to diffuse into tumor from
blood vessels, or the high concentration of HA-Pt(IV) nanoparticles, sustainably eluted from
the injection depot, engulfed by the infiltrating macrophages via phagocytosis, and
subsequently transported to the neighboring tumor cells. It is not surprising that the self-
regulated uptake of Pt(IV) molecules and the simultaneous recruitment of macrophages
could work synergistically to result in a superior tumor suppression compared to the
intravenous cisplatin. Further investigations on the identity of the infiltrating macrophages
using M1/M2 specific markers in the presence of fluorescently labeled, intratumorally
ACS Paragon Plus Environment

Page 17 of 25
Molecular Pharmaceutics
1
2
3
4
5
administered HA-Pt(IV) may elucidate the dynamics of the tumor environment and the
immune cell activation.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 7. Mouse AT84 tumors treated with either intravenous cisplatin or intratumoral
HA-Pt(IV), and stained with DAPI, CD8a, CD11b and CD11c.
3.4 RNA sequencing
In the gene expression analysis, 35 genes in the HA-Pt(IV)-treated were filtered as
differentially expressed compared to the untreated control, 20 of which were
downregulated, whereas 15 of which were upregulated (p<0.1). If the analysis threshold
was tightened to p<0.05, 17 genes in the HA-Pt(IV)-treated were filtered as differentially
expressed compared to the untreated control. Nine of the 17 were downregulated, and the
remainder of 8 were upregulated. A complete list of differential gene expression analysis
result is included in the supplementary data.
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 18 of 25
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 8. Volcano plot of the gene expression analysis.
The 3 most over-expressed genes in HA-Pt(IV)-treated were GALTN2l, Rps13-ps2, and Nnt;
and the 3 most under-expressed genes in the HA-Pt(IV)-treated were EGF, Rps16-ps2, and
Dpp10. The highly expressed GALTN2l gene encodes an enzyme, N-
acetylgalactosaminyltransferase 2-like that belongs to a family of over 20 polypeptides of
N-acetylgalactosaminyltransferases. This group of transferases catalyzes the transfer of N-
acetylgalactosamine from uridine diphosphate N-acetylglucosamine to the hydroxyl group
of a serine or threonine residue 59. The overexpression of GALTN2 has been linked to the
inhibition of neuroblastoma proliferation 60, whereas the underexpression of this gene has
been associated with the proliferation, invasion and tumor metastasis of gastric
adenocarcinoma 61. In the current study, the upregulation of GALTN2l in HA-Pt(IV)-treated
tumors may partially contribute to the superior tumor suppression and the resultant,
improved anti-cancer effectiveness. On the other hand, the underexpressed EGF gene
encodes epidermal growth factor protein, which stimulates cell proliferation by binding to
the epidermal growth factor receptor. Overexpression of EGF has been correlated with
progression and invasiveness of breast cancer 62. In this analysis, the EGF expression shows
ACS Paragon Plus Environment

Page 19 of 25
Molecular Pharmaceutics
1
2
3
4
5
6
significant downregulation in HA-Pt(IV)-treated tumors compared to the untreated control.
The underexpression of EGF may explain the prolonged survival and the enhanced tumor
suppression observed in the drug treated HNCs compared to the untreated tumors.
7
8
9
Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment
analysis are two of the common downstream procedures to interpret the differential gene
expression results in a biological context. GO enrichment analysis effectively categorizes
differential gene expressions into specific biological processes. KEGG uses molecular-level
information generated from large-scale, high-throughput gene datasets to help elucidate
high-level functions and utilities of the biological system 63.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In the GO analysis, the 3 most significantly enriched GO categories for the HA-Pt(IV)
treatment group are: 1) nucleoside diphosphate kinase activity, 2) piRNA metabolic
process, and 3) intrinsic component of nuclear inner membrane, which belong to the
categories of molecular function-kinase activity, biological process-RNA metabolic process,
and cellular component-nuclear membrane, respectively. The 3 most significantly enriched
GO categories for the untreated group are: 1) positive regulation of interleukin 12
production, 2) cargo receptor activity, and 3) toxin metabolic process, which belong to the
categories of biological process-cytokine production, molecular function-cargo receptor
activity, and biological process-cellular metabolic process, respectively.
In the KEGG analysis, 89 pathways were enriched and analyzed to understand the
biological roles of the differentially expressed genes. In the HA-Pt(IV) treated group, the 5
most important KEGG pathways include: 1) pentose and glucuronate interconversions, 2)
starch and sucrose metabolism, 3) ribosome, 4) porphyrin and chlorophyll metabolism,
and 5) galactose metabolism (all with p<0.037, and 17 to 83 gene sets). It is worth noting
that 4 out of these 5 pathways are associated with metabolism – 3 are identified in
carbohydrate metabolism; and 1 is identified in cofactor/vitamin metabolism, and 1 of 5 is
associated with translation. Carbohydrate metabolism is commonly dysregulated in
cancers, especially the pentose and glucuronate interconversions 64, and changes in the
metabolism may reflect a response to the treatment.
The 5 most important KEGG pathways for the control group include: 1) allograft rejection,
2) leishmania infection, 3) autoimmune thyroid disease, 4) intestinal immune network for
IgA production, and 5) primary immunodeficiency (all with p<0.033, and 28 to 62 gene
sets). Different from the HA-Pt(IV) treated group, none of the 5 most important pathways
involved in the control group are associated with metabolism. Instead, 4 of the 5 most
important pathways are associated with immune disease and 1 is associated with
infectious disease. The close correlation between the untreated HNCs and the KEGG
immune disease pathways may suggest that HNC is not a localized disease and the immune
system may be highly involved in its progression. Therefore, non-traditional therapies such
as ICD-eliciting molecules (e.g. HA-Pt(IV)), immunomodulators (e.g. TLR agonists) 65, 66, and
immune checkpoint inhibitors (e.g. anti-PD1 antibodies) 65, 67 may be more targeted,
effective solutions for treating HNCs compared to conventional therapies that do not
activate the immune system.
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 20 of 25
1
2
3
4
Conclusion
5
6
7
8
In an effort to understand the indications and limitations of evolving platinum
chemotherapy, we reported the synthesis, characterization and preclinical testing of a
platinum(IV)-based, ICD-eliciting, and carrier-delivered nanoconjugate for potential
oncology applications. This study showed for the first time that intralesional Pt(IV) induced
a form of ICD in head and neck cancers. In addition, immunological evaluation of the tumors
demonstrated increased lymphocyte and macrophage infiltration compared to the
intravenous cisplatin treatment. HA-Pt(IV) may also find utility in concurrent chemo-
radiation therapy, making practical and effective use of the radiation-sensitizing platinum
and the immune responsive radiotherapy. We are currently conducting a clinical study
combining intralesional HA-Pt(IV) and radiation therapy in pet dogs with naturally-
occurring, externally-accessible malignant tumors at the Kansas State University Veterinary
Health Center. Although platinum drugs were discovered over a century ago and have been
used in oncology for more than four decades, platinum therapies still offer new interesting
avenues for improved treatments.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Acknowledgements
Partial financial support was provided by HylaPharm LLC. RL and LF were partially
supported by the National Cancer Institute R01-CA173292 and generous gifts from the
Brandmeyer family and Hodgdon family foundations. The KU Genome Sequencing Core and
KU NMR labs were supported by NIH grants P20GM103638 and P50GM069663,
respectively.
Supporting Information
The supporting information of this study is available online.
 Nuclear Magnetic Resonance (NMR) spectrums of HA-Pt(IV) and intermediates.
 Individual tumor growth curves.
 RNA sequencing metadata and significant differentially expressed genes.
 Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO)
significant pathways analysis.
Reference
1.
diagnosis. Oral Maxillofac Surg Clin North Am 2014, 26, (2), 123-41.
2. https://www.cancer.gov/types/head-and-neck/head-neck-fact-sheet. National Cancer Institute,
Accessed on April 11, 2019.
Vigneswaran, N.; Williams, M. D. Epidemiologic trends in head and neck cancer and aids in
3.
Fessas, P.; Lee, H.; Ikemizu, S.; Janowitz, T. A molecular and preclinical comparison of the PD-1-
targeted T-cell checkpoint inhibitors nivolumab and pembrolizumab. Semin Oncol 2017, 44, (2), 136-140.
ACS Paragon Plus Environment

Page 21 of 25
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
4.
Cai, S.; Xie, Y.; Bagby, T. R.; Cohen, M. S.; Forrest, M. L. Intralymphatic chemotherapy using a
hyaluronan-cisplatin conjugate. The Journal of surgical research 2008, 147, (2), 247-52.
5. Cai, S.; Xie, Y.; Davies, N. M.; Cohen, M. S.; Forrest, M. L. Pharmacokinetics and disposition of a
localized lymphatic polymeric hyaluronan conjugate of cisplatin in rodents. Journal of pharmaceutical
sciences 2010, 99, (6), 2664-71.
6.
Xie, Y.; Aillon, K. L.; Cai, S.; Christian, J. M.; Davies, N. M.; Berkland, C. J.; Forrest, M. L.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Pulmonary delivery of cisplatin-hyaluronan conjugates via endotracheal instillation for the treatment of
lung cancer. International journal of pharmaceutics 2010, 392, (1-2), 156-63.
7.
Zhang, T.; Yang, Q.; Forrest, W. C.; Cai, S.; Aires, D.; Forrest, M. L. A Lanthanum-Tagged
Chemotherapeutic Agent HA-Pt to Track the In Vivo Distribution of Hyaluronic Acid Complexes. BAOJ
Pharm Sci 2015, 1, (1).
8.
Zhang, T.; Cai, S.; Groer, C.; Forrest, W. C.; Yang, Q.; Mohr, E.; Douglas, J.; Aires, D.; Axiak-
Bechtel, S. M.; Selting, K. A.; Swarz, J. A.; Tate, D. J.; Bryan, J. N.; Forrest, M. L. Hyaluronan-Lysine
Cisplatin Drug Carrier for Treatment of Localized Cancers: Pharmacokinetics, Tolerability, and Efficacy in
Rodents and Canines. J Pharm Sci 2016, 105, (6), 1891-1900.
9.
Ishiguro, S.; Cai, S.; Uppalapati, D.; Turner, K.; Zhang, T.; Forrest, W. C.; Forrest, M. L.; Tamura,
M. Intratracheal Administration of Hyaluronan-Cisplatin Conjugate Nanoparticles Significantly
Attenuates Lung Cancer Growth in Mice. Pharmaceutical research 2016, 33, (10), 2517-29.
10.
Venable, R. O.; Worley, D. R.; Gustafson, D. L.; Hansen, R. J.; Ehrhart, E. J., 3rd; Cai, S.; Cohen, M.
S.; Forrest, M. L. Effects of intratumoral administration of a hyaluronan-cisplatin nanoconjugate to five
dogs with soft tissue sarcomas. American journal of veterinary research 2012, 73, (12), 1969-76.
11.
Cai, S.; Xie, Y.; Davies, N. M.; Cohen, M. S.; Forrest, M. L. Carrier-based intralymphatic cisplatin
chemotherapy for the treatment of metastatic squamous cell carcinoma of the head & neck.
Therapeutic delivery 2010, 1, (2), 237-45.
12.
Yang, Q.; Cui, H.; Cai, S.; Yang, X.; Forrest, M. L. In vivo photoacoustic imaging of chemotherapy-
induced apoptosis in squamous cell carcinoma using a near-infrared caspase-9 probe. J Biomed Opt
2011, 16, (11), 116026.
13.
Cohen, S. M.; Rockefeller, N.; Mukerji, R.; Durham, D.; Forrest, M. L.; Cai, S.; Cohen, M. S.;
Shnayder, Y. Efficacy and toxicity of peritumoral delivery of nanoconjugated cisplatin in an in vivo
murine model of head and neck squamous cell carcinoma. JAMA otolaryngology-- head & neck surgery
2013, 139, (4), 382-7.
14.
Cai, S.; Zhang, T.; Forrest, W. C.; Yang, Q.; Groer, C.; Mohr, E.; Aires, D. J.; Axiak-Bechtel, S. M.;
Flesner, B. K.; Henry, C. J.; Selting, K. A.; Tate, D.; Swarz, J. A.; Bryan, J. N.; Forrest, M. L. Phase I-II clinical
trial of hyaluronan-cisplatin nanoconjugate in dogs with naturally occurring malignant tumors. American
journal of veterinary research 2016, 77, (9), 1005-16.
15.
Cai, S.; Zhang, T.; Groer, C.; Forrest, M.; Aires, D.; Otte, V.; Barchman, S.; Faerber, A.; Forrest, M.
L. Injectable Chemotherapy Downstaged Oral Squamous Cell Carcinoma from Nonresectable to
Resectable in a Rescue Dog: Diagnosis, Treatment, and Outcome. Case Rep Vet Med 2018, 2018,
9078537.
16.
Zhang, J. Z.; Bonnitcha, P.; Wexselblatt, E.; Klein, A. V.; Najajreh, Y.; Gibson, D.; Hambley, T. W.
Facile preparation of mono-, di- and mixed-carboxylato platinum(IV) complexes for versatile anticancer
prodrug design. Chemistry 2013, 19, (5), 1672-6.
17.
Wexselblatt, E.; Yavin, E.; Gibson, D. Platinum(IV) prodrugs with haloacetato ligands in the axial
positions can undergo hydrolysis under biologically relevant conditions. Angew Chem Int Ed Engl 2013,
52, (23), 6059-62.
18.
Galanski, M.; Keppler, B. K. Synthesis and Characterization of New Ethylenediamine
Platinum(IV) Complexes Containing Lipophilic Carboxylate Ligands. Met Based Drugs 1995, 2, (1), 57-63.
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 22 of 25
1
2
3
4
5
6
7
8
9
19.
Ma, J.; Wang, Q.; Yang, X.; Hao, W.; Huang, Z.; Zhang, J.; Wang, X.; Wang, P. G. Glycosylated
platinum(iv) prodrugs demonstrated significant therapeutic efficacy in cancer cells and minimized side-
effects. Dalton Trans 2016, 45, (29), 11830-8.
20.
B. K.; Koellensperger, G. The impact of whole human blood on the kinetic inertness of platinum(iv)
prodrugs - an HPLC-ICP-MS study. Dalton Trans 2018, 47, (15), 5252-5258.
Theiner, S.; Grabarics, M.; Galvez, L.; Varbanov, H. P.; Sommerfeld, N. S.; Galanski, M.; Keppler,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
21.
platinum(IV) complexes in ovarian cancer cell lines. J Inorg Biochem 2004, 98, (10), 1614-24.
22. Chen, C. K.; Zhang, J. Z.; Aitken, J. B.; Hambley, T. W. Influence of equatorial and axial
carboxylato ligands on the kinetic inertness of platinum(IV) complexes in the presence of ascorbate and
cysteine and within DLD-1 cancer cells. J Med Chem 2013, 56, (21), 8757-64.
Hall, M. D.; Amjadi, S.; Zhang, M.; Beale, P. J.; Hambley, T. W. The mechanism of action of
23.
Sabbatini, M.; Zanellato, I.; Ravera, M.; Gabano, E.; Perin, E.; Rangone, B.; Osella, D. Pt(IV)
Bifunctional Prodrug Containing 2-(2-Propynyl)octanoato Axial Ligand: Induction of Immunogenic Cell
Death on Colon Cancer. J Med Chem 2019, 62, (7), 3395-3406.
24.
Hato, S. V.; Khong, A.; de Vries, I. J.; Lesterhuis, W. J. Molecular pathways: the immunogenic
effects of platinum-based chemotherapeutics. Clinical cancer research : an official journal of the
American Association for Cancer Research 2014, 20, (11), 2831-7.
25.
Kroemer, G.; Galluzzi, L.; Kepp, O.; Zitvogel, L. Immunogenic cell death in cancer therapy. Annu
Rev Immunol 2013, 31, 51-72.
26.
Tesniere, A.; Schlemmer, F.; Boige, V.; Kepp, O.; Martins, I.; Ghiringhelli, F.; Aymeric, L.;
Michaud, M.; Apetoh, L.; Barault, L.; Mendiboure, J.; Pignon, J. P.; Jooste, V.; van Endert, P.; Ducreux, M.;
Zitvogel, L.; Piard, F.; Kroemer, G. Immunogenic death of colon cancer cells treated with oxaliplatin.
Oncogene 2010, 29, (4), 482-91.
27.
complexes for dual-targeting anticancer therapy. Chem Commun (Camb) 2014, 50, (19), 2465-8.
28. Zhang, T.; Cai, S.; Forrest, W. C.; Mohr, E.; Yang, Q.; Forrest, M. L. Development and Validation
Suntharalingam, K.; Song, Y.; Lippard, S. J. Conjugation of vitamin E analog alpha-TOS to Pt(IV)
of an Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Method for Quantitative Analysis of
Platinum in Plasma, Urine, and Tissues. Appl Spectrosc 2016, 70, (9), 1529-36.
29.
immunotherapy of head and neck squamous cell carcinoma. The Laryngoscope 1995, 105, (10), 1077-80.
30. Paolini, F.; Massa, S.; Manni, I.; Franconi, R.; Venuti, A. Immunotherapy in new pre-clinical
models of HPV-associated oral cancers. Hum Vaccin Immunother 2013, 9, (3), 534-43.
31. Golden, E. B.; Frances, D.; Pellicciotta, I.; Demaria, S.; Helen Barcellos-Hoff, M.; Formenti, S. C.
Hier, M. P.; Black, M. J.; Shenouda, G.; Sadeghi, N.; Karp, S. E. A murine model for the
Radiation fosters dose-dependent and chemotherapy-induced immunogenic cell death.
Oncoimmunology 2014, 3, e28518.
32.
mice. Cancer Chemother Pharmacol 1989, 24, (3), 148-54.
33. Andrews, S. FastQC: A Quality Control Tool for High Throughput Sequence Data.
http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ (5/14/2020),
34. Patro, R.; Duggal, G.; Love, M. I.; Irizarry, R. A.; Kingsford, C. Salmon provides fast and bias-
aware quantification of transcript expression. Nature Methods 2017, 14, (4), 417-419.
35. Genome Reference Consortium Mouse Build 38.
https://www.ncbi.nlm.nih.gov/assembly/GCF_000001635.20/ (5/14/2020),
36. Soneson, C.; Love, M. I.; Robinson, M. D. Differential analyses for RNA-seq: transcript-level
estimates improve gene-level inferences. F1000Research 2015, 4, 1521.
37. Subramanian, A.; Tamayo, P.; Mootha, V. K.; Mukherjee, S.; Ebert, B. L.; Gillette, M. A.;
Tomayko, M. M.; Reynolds, C. P. Determination of subcutaneous tumor size in athymic (nude)
Paulovich, A.; Pomeroy, S. L.; Golub, T. R.; Lander, E. S.; Mesirov, J. P. Gene set enrichment analysis: a
ACS Paragon Plus Environment

Page 23 of 25
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A
2005, 102, (43), 15545-50.
38.
Mootha, V. K.; Lindgren, C. M.; Eriksson, K. F.; Subramanian, A.; Sihag, S.; Lehar, J.; Puigserver,
P.; Carlsson, E.; Ridderstrale, M.; Laurila, E.; Houstis, N.; Daly, M. J.; Patterson, N.; Mesirov, J. P.; Golub,
T. R.; Tamayo, P.; Spiegelman, B.; Lander, E. S.; Hirschhorn, J. N.; Altshuler, D.; Groop, L. C. PGC-1alpha-
responsive genes involved in oxidative phosphorylation are coordinately downregulated in human
diabetes. Nat Genet 2003, 34, (3), 267-73.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
39.
Zhang, J. Z.; Wexselblatt, E.; Hambley, T. W.; Gibson, D. Pt(IV) analogs of oxaliplatin that do not
follow the expected correlation between electrochemical reduction potential and rate of reduction by
ascorbate. Chem Commun (Camb) 2012, 48, (6), 847-9.
40.
Pt(IV) prodrugs designed to bind non-covalently to human serum albumin for drug delivery. J Am Chem
Soc 2014, 136, (24), 8790-8.
41.
conjugate as a reduction-responsive carrier for triggered drug release. J. Mater. Chem. B 2015, 3, 8203-
8211.
Zheng, Y. R.; Suntharalingam, K.; Johnstone, T. C.; Yoo, H.; Lin, W.; Brooks, J. G.; Lippard, S. J.
He, S.; Cong, Y.; Zhou, D.; Li, J.; Xie, Z.; Chen, X.; Jing, X.; Huang, Y. A dextran-platinum(IV)
42.
Ling, X.; Shen, Y.; Sun, R.; Zhang, M.; Li, C.; Mao, J.; Xing, J.; Sun, C.; Tu, J. Tumor-targeting
delivery of hyaluronic acid-platinum(IV) nanoconjugate to reduce toxicity and improve survival. Polym
Chem 2015, 6, 1541-1552.
43.
blood cells in vitro. Cancer Chemother Pharmacol 2002, 50, (1), 9-15.
44. Sternberg, C. N.; Petrylak, D. P.; Sartor, O.; Witjes, J. A.; Demkow, T.; Ferrero, J. M.; Eymard, J. C.;
Falcon, S.; Calabro, F.; James, N.; Bodrogi, I.; Harper, P.; Wirth, M.; Berry, W.; Petrone, M. E.; McKearn,
T. J.; Noursalehi, M.; George, M.; Rozencweig, M. Multinational, double-blind, phase III study of
prednisone and either satraplatin or placebo in patients with castrate-refractory prostate cancer
progressing after prior chemotherapy: the SPARC trial. J Clin Oncol 2009, 27, (32), 5431-8.
Carr, J. L.; Tingle, M. D.; McKeage, M. J. Rapid biotransformation of satraplatin by human red
45.
Cohen, S. M.; Mukerji, R.; Cai, S.; Damjanov, I.; Forrest, M. L.; Cohen, M. S. Subcutaneous
delivery of nanoconjugated doxorubicin and cisplatin for locally advanced breast cancer demonstrates
improved efficacy and decreased toxicity at lower doses than standard systemic combination therapy in
vivo. American journal of surgery 2011, 202, (6), 646-52; discussion 652-3.
46.
Yang, Q.; Aires, D. J.; Cai, S.; Fraga, G. R.; Zhang, D.; Li, C. Z.; Forrest, M. L. In vivo efficacy of
nano hyaluronan-conjugated cisplatin for treatment of murine melanoma. Journal of drugs in
dermatology : JDD 2014, 13, (3), 283-7.
47.
for cisplatin therapy in breast cancer with improved tumor efficacy and lower systemic toxicity in vivo.
American journal of surgery 2009, 198, (6), 781-6.
48.
lymphatic drainage of a biopolymer-based imaging agent. Pharmaceutics 2012, 4, (2), 276-95.
49. Lovgren, T.; Sarhan, D.; Truxova, I.; Choudhary, B.; Maas, R.; Melief, J.; Nystrom, M.; Edback, U.;
Vermeij, R.; Scurti, G.; Nishimura, M.; Masucci, G.; Karlsson-Parra, A.; Lundqvist, A.; Adamson, L.;
Kiessling, R. Enhanced stimulation of human tumor-specific T cells by dendritic cells matured in the
presence of interferon-gamma and multiple toll-like receptor agonists. Cancer Immunol Immunother
2017, 66, (10), 1333-1344.
Cohen, M. S.; Cai, S.; Xie, Y.; Forrest, M. L. A novel intralymphatic nanocarrier delivery system
Bagby, T. R.; Cai, S.; Duan, S.; Thati, S.; Aires, D. J.; Forrest, L. Impact of molecular weight on
50.
Casares, N.; Pequignot, M. O.; Tesniere, A.; Ghiringhelli, F.; Roux, S.; Chaput, N.; Schmitt, E.;
Hamai, A.; Hervas-Stubbs, S.; Obeid, M.; Coutant, F.; Metivier, D.; Pichard, E.; Aucouturier, P.; Pierron,
G.; Garrido, C.; Zitvogel, L.; Kroemer, G. Caspase-dependent immunogenicity of doxorubicin-induced
tumor cell death. J Exp Med 2005, 202, (12), 1691-701.
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 24 of 25
1
2
3
4
5
6
7
8
9
51.
Pfirschke, C.; Engblom, C.; Rickelt, S.; Cortez-Retamozo, V.; Garris, C.; Pucci, F.; Yamazaki, T.;
Poirier-Colame, V.; Newton, A.; Redouane, Y.; Lin, Y. J.; Wojtkiewicz, G.; Iwamoto, Y.; Mino-Kenudson,
M.; Huynh, T. G.; Hynes, R. O.; Freeman, G. J.; Kroemer, G.; Zitvogel, L.; Weissleder, R.; Pittet, M. J.
Immunogenic Chemotherapy Sensitizes Tumors to Checkpoint Blockade Therapy. Immunity 2016, 44,
(2), 343-54.
52.
Kepp, O.; Senovilla, L.; Vitale, I.; Vacchelli, E.; Adjemian, S.; Agostinis, P.; Apetoh, L.; Aranda, F.;
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Barnaba, V.; Bloy, N.; Bracci, L.; Breckpot, K.; Brough, D.; Buque, A.; Castro, M. G.; Cirone, M.; Colombo,
M. I.; Cremer, I.; Demaria, S.; Dini, L.; Eliopoulos, A. G.; Faggioni, A.; Formenti, S. C.; Fucikova, J.;
Gabriele, L.; Gaipl, U. S.; Galon, J.; Garg, A.; Ghiringhelli, F.; Giese, N. A.; Guo, Z. S.; Hemminki, A.;
Herrmann, M.; Hodge, J. W.; Holdenrieder, S.; Honeychurch, J.; Hu, H. M.; Huang, X.; Illidge, T. M.; Kono,
K.; Korbelik, M.; Krysko, D. V.; Loi, S.; Lowenstein, P. R.; Lugli, E.; Ma, Y.; Madeo, F.; Manfredi, A. A.;
Martins, I.; Mavilio, D.; Menger, L.; Merendino, N.; Michaud, M.; Mignot, G.; Mossman, K. L.; Multhoff,
G.; Oehler, R.; Palombo, F.; Panaretakis, T.; Pol, J.; Proietti, E.; Ricci, J. E.; Riganti, C.; Rovere-Querini, P.;
Rubartelli, A.; Sistigu, A.; Smyth, M. J.; Sonnemann, J.; Spisek, R.; Stagg, J.; Sukkurwala, A. Q.; Tartour, E.;
Thorburn, A.; Thorne, S. H.; Vandenabeele, P.; Velotti, F.; Workenhe, S. T.; Yang, H.; Zong, W. X.;
Zitvogel, L.; Kroemer, G.; Galluzzi, L. Consensus guidelines for the detection of immunogenic cell death.
Oncoimmunology 2014, 3, (9), e955691.
53.
Martins, I.; Kepp, O.; Schlemmer, F.; Adjemian, S.; Tailler, M.; Shen, S.; Michaud, M.; Menger, L.;
Gdoura, A.; Tajeddine, N.; Tesniere, A.; Zitvogel, L.; Kroemer, G. Restoration of the immunogenicity of
cisplatin-induced cancer cell death by endoplasmic reticulum stress. Oncogene 2011, 30, (10), 1147-58.
54.
Hughes, R.; Qian, B. Z.; Rowan, C.; Muthana, M.; Keklikoglou, I.; Olson, O. C.; Tazzyman, S.;
Danson, S.; Addison, C.; Clemons, M.; Gonzalez-Angulo, A. M.; Joyce, J. A.; De Palma, M.; Pollard, J. W.;
Lewis, C. E. Perivascular M2 Macrophages Stimulate Tumor Relapse after Chemotherapy. Cancer Res
2015, 75, (17), 3479-91.
55.
DeNardo, D. G.; Brennan, D. J.; Rexhepaj, E.; Ruffell, B.; Shiao, S. L.; Madden, S. F.; Gallagher, W.
M.; Wadhwani, N.; Keil, S. D.; Junaid, S. A.; Rugo, H. S.; Hwang, E. S.; Jirstrom, K.; West, B. L.; Coussens, L.
M. Leukocyte complexity predicts breast cancer survival and functionally regulates response to
chemotherapy. Cancer Discov 2011, 1, (1), 54-67.
56.
Chen, L.; Li, J.; Wang, F.; Dai, C.; Wu, F.; Liu, X.; Li, T.; Glauben, R.; Zhang, Y.; Nie, G.; He, Y.; Qin,
Z. Tie2 Expression on Macrophages Is Required for Blood Vessel Reconstruction and Tumor Relapse
after Chemotherapy. Cancer Res 2016, 76, (23), 6828-6838.
57.
Zhao, Y.; Zhang, C.; Gao, L.; Yu, X.; Lai, J.; Lu, D.; Bao, R.; Wang, Y.; Jia, B.; Wang, F.; Liu, Z.
Chemotherapy-Induced Macrophage Infiltration into Tumors Enhances Nanographene-Based
Photodynamic Therapy. Cancer Res 2017, 77, (21), 6021-6032.
58.
cancer. Immunotherapy 2013, 5, (10), 1075-87.
59. Hussain, M. R.; Hoessli, D. C.; Fang, M. N-acetylgalactosaminyltransferases in cancer.
Oncotarget 2016, 7, (33), 54067-54081.
60. Ho, W. L.; Chou, C. H.; Jeng, Y. M.; Lu, M. Y.; Yang, Y. L.; Jou, S. T.; Lin, D. T.; Chang, H. H.; Lin, K.
Panni, R. Z.; Linehan, D. C.; DeNardo, D. G. Targeting tumor-infiltrating macrophages to combat
H.; Hsu, W. M.; Huang, M. C. GALNT2 suppresses malignant phenotypes through IGF-1 receptor and
predicts favorable prognosis in neuroblastoma. Oncotarget 2014, 5, (23), 12247-59.
61.
Liu, S. Y.; Shun, C. T.; Hung, K. Y.; Juan, H. F.; Hsu, C. L.; Huang, M. C.; Lai, I. R. Mucin
glycosylating enzyme GALNT2 suppresses malignancy in gastric adenocarcinoma by reducing MET
phosphorylation. Oncotarget 2016, 7, (10), 11251-62.
62.
Metastasis Rev 1993, 12, (3-4), 255-74.
63. Caly, L.; Druce, J. D.; Catton, M. G.; Jans, D. A.; Wagstaff, K. M. The FDA-approved drug
Khazaie, K.; Schirrmacher, V.; Lichtner, R. B. EGF receptor in neoplasia and metastasis. Cancer
ivermectin inhibits the replication of SARS-CoV-2 in vitro. Antiviral Res 2020, 178, 104787.
ACS Paragon Plus Environment

Page 25 of 25
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
64.
Rosario, S. R.; Long, M. D.; Affronti, H. C.; Rowsam, A. M.; Eng, K. H.; Smiraglia, D. J. Pan-cancer
analysis of transcriptional metabolic dysregulation using The Cancer Genome Atlas. Nat Commun 2018,
9, (1), 5330.
65.
M.; Messer, K. S.; Guiducci, C.; Coffman, R. L.; Kitaura, K.; Matsutani, T.; Suzuki, R.; Carson, D. A.;
Hayashi, T.; Cohen, E. E. Combination immunotherapy with TLR agonists and checkpoint inhibitors
suppresses head and neck cancer. JCI Insight 2017, 2, (18).
Sato-Kaneko, F.; Yao, S.; Ahmadi, A.; Zhang, S. S.; Hosoya, T.; Kaneda, M. M.; Varner, J. A.; Pu,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
66.
toll-like receptor 9 (TLR-9) agonist: from bench to bedside. Crit Rev Oncol Hematol 2015, 94, (1), 31-44.
67. Mandal, R.; Senbabaoglu, Y.; Desrichard, A.; Havel, J. J.; Dalin, M. G.; Riaz, N.; Lee, K. W.; Ganly,
I.; Hakimi, A. A.; Chan, T. A.; Morris, L. G. The head and neck cancer immune landscape and its
immunotherapeutic implications. JCI Insight 2016, 1, (17), e89829.
Wittig, B.; Schmidt, M.; Scheithauer, W.; Schmoll, H. J. MGN1703, an immunomodulator and
ACS Paragon Plus Environment