cells
ArticleA New Synthesized Dicarboxylated Oxy-Heparin Efficiently
Attenuates Tumor Growth and Metastasis
Li Li 1 , Uri Barash 2 , Neta Ilan 2 , Malik Farhoud 2 , Xiao Zhang 3 , Israel Vlodavsky 2, * and Jin-Ping Li 4, *
1
2

3
4

*

Citation: Li, L.; Barash, U.; Ilan, N.;
Farhoud, M.; Zhang, X.; Vlodavsky, I.;
Li, J.-P. A New Synthesized
Dicarboxylated Oxy-Heparin

Shenzhen Hepalink Pharmaceutical Group Co., Ltd., Shenzhen 518057, China; lili@hepalink.com
Technion Integrated Cancer Center, Rappaport Faculty of Medicine, Technion, Haifa 3525422, Israel;ubarash@yahoo.com (U.B.); netailan@technion.ac.il (N.I.); malik.far.96@gmail.com (M.F.)
Department of Medical Cell Biology, Uppsala University, 751 23 Uppsala, Sweden; xiao.zhang@uu.se
Department of Medical Biochemistry and Microbiology, SciLifeLab, Uppsala University,
751 23 Uppsala, Sweden
Correspondence: vlodavsk@mail.huji.ac.il (I.V.); jin-ping.li@imbim.uu.se (J.-P.L.)

Abstract: Heparanase (Hpa1) is expressed by tumor cells and cells of the tumor microenvironment and functions to remodel the extracellular matrix (ECM) and regulate the bioavailability ofECM-bound factors that support tumor growth. Heparanase expression is upregulated in humancarcinomas, sarcomas, and hematological malignancies, correlating with increased tumor metastasis,
vascular density, and shorter postoperative survival of cancer patients, and encouraging the development of heparanase inhibitors as anti-cancer drugs. Among these are heparin/HS mimetics, theonly heparanase-inhibiting compounds that are being evaluated in clinical trials. We have synthesized dicarboxylated oxy-heparins (DCoxHs) containing three carboxylate groups per split residue(DC-Hep). The resulting lead compound (termed XII) was upscaled, characterized, and examined for
its effectiveness in tumor models. Potent anti-tumorigenic effects were obtained in models of pancreatic carcinoma, breast cancer, mesothelioma, and myeloma, yielding tumor growth inhibition (TGI)
values ranging from 21 to 70% and extending the survival time of the mice. Of particular significancewas the inhibition of spontaneous metastasis in an orthotopic model of breast carcinoma following
resection of the primary tumor. It appears that apart from inhibition of heparanase enzymatic activity,
compound XII reduces the levels of heparanase protein and inhibits its cellular uptake and activation.
Heparanase-dependent and -independent effects of XII are being investigated. Collectively, ourpre-clinical studies with compound XII strongly justify its examination in cancer patients.

Efficiently Attenuates Tumor Growthand Metastasis. Cells 2024, 13, 211.
https://doi.org/10.3390/

Keywords: heparanase; primary tumor growth; metastasis; tumor growth inhibition; mesothelioma;
breast carcinoma; pancreatic carcinoma; chemically modified heparincells13030211
Academic Editor: Edward V.
Prochownik
Received: 30 November 2023
Revised: 12 January 2024
Accepted: 18 January 2024
Published: 23 January 2024

Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction
Heparanase is an endoglucuronidase that cleaves heparan sulfate (HS), thereby altering the structure and function of heparan sulfate proteoglycans (HSPG) and contributing totumor-mediated remodeling of the cell surface and extracellular matrix (ECM) [1–3]. These
actions dynamically impact multiple pro-tumorigenic regulatory pathways, most notably
by augmenting the bioavailability of growth factors and cytokines bound to heparan sulfate
in the tumor microenvironment [2,4–8]. In addition, heparanase upregulates the expression of multiple genes (i.e., VEGF, HGF, RANKL, MMP-9, IL-2, and IFN-γ) that promote
aggressive tumor progression and inflammation [9]. Intense research efforts during the lasttwo decades revealed that heparanase expression is upregulated in various human carcinomas, sarcomas, and hematological malignancies [4–6,8,10]. Compelling evidence ties
heparanase to all steps of tumor formation, including tumor initiation, growth, metastasis,and chemoresistance [11–13]. Likewise, many clinical association studies have consistently
demonstrated that upregulation of heparanase expression correlates with increased tumor
size, tumor angiogenesis, enhanced metastasis, and poor prognosis [14–17], providing

Cells 2024, 13, 211. https://doi.org/10.3390/cells13030211

https://www.mdpi.com/journal/cells

Cells 2024, 13, 211

2 of 18strong support for the pro-tumorigenic function of the enzyme. By contrast, knockdown ofheparanase or treatments of tumor-bearing mice with heparanase-inhibiting compounds
markedly attenuate tumor progression, underscoring the potential of anti-heparanase
therapy for multiple types of cancer and encouraging the development of heparanase
inhibitors as anti-cancer drugs [12,14]. Heparin/HS-mimetics (i.e., Muparfostat = PI-88,
Roneparstat = SST0001, Necuparanib = M402, Pixatimod = PG545) that inhibit heparanase
enzymatic activity were and are being evaluated in clinical trials for various types of cancer
and appear to be well tolerated [18–21]. Heparanase-inhibiting small molecules [22] andheparanase-neutralizing monoclonal antibodies were developed, in part based on the crystal structure of the heparanase protein [23], and are being examined in pre-clinical studies.
Heparin, exclusively produced by mast cells, is a highly sulfated form of HS. Early
studies evidenced unfractionated heparin (UFH) as a substrate and potent heparanase
inhibitor [24]. In subsequent studies, investigations were mainly oriented to generate
non-anticoagulant heparin derivatives to overcome the anticoagulation and bleedingside effects and to improve the pharmacokinetics and bioavailability of newly generated
species of heparin. Among these compounds is Roneparstat (=SST0001), a chemicallymodified 100% N-desulphated, N-reacetylated, and 25% glycol-split heparin with very
low anticoagulant activity and a molecular weight ranging from 15,000 to 25,000 Da [25].
Roneparstat was highly effective in murine models of myeloma [12]. Based on thispreclinical evidence, a multicenter, phase I study was performed to assess the safety
and tolerability profile of Roneparstat in patients with relapsed/refractory myeloma(clinicaltrials.gov identifier: NCT01764880). The compound exhibited excellent safety andtolerability profiles and could be administered at dose levels of 200 and 400 mg/patient/day
without causing any clinically relevant toxicity [18]. It was concluded that even though thephase I study did not provide evidence of a direct anti-myeloma effect of Ronepartstat inhumans, exploration of Roneparstat in combination regimens for the treatment of myeloma,
and possibly other types of cancer, was justified [21]. Of note, a recent phase 1b clinical trial(PG545 = Pixatimod in combination with nivolumab = anti-PD-1 drug) showed signs ofclinical benefit for colorectal patients [19].
To increase the affinity of glycol-split residues for the active site of heparanase andimprove the pharmacokinetic properties of Roneparstat, dicarboxylated oxy-heparins(DCoxHs) containing three carboxylate groups per split residue (DC-Hep) were prepared [25]. The resulting lead compound (XII) was selected for upscaling, characterization,and examination in mouse tumor models toward further development and investigationof its mode of action. Promising results were obtained in models of pancreatic carcinoma,
breast cancer, mesothelioma, and myeloma as a single agent, yielding tumor growth inhibition (TGI) values ranging from 21% to 70%. Of particular significance was the inhibitionof spontaneous metastasis in an orthotopic model of breast carcinoma following resectionof the primary tumor. It appears that apart from inhibition of heparanase expression
levels and enzymatic activity, compound XII competes with HS on the cell surface andthereby inhibits cellular binding, uptake, and processing/activation of the secreted 65 kDa
latent form of the enzyme. Collectively, our pre-clinical studies with compound XII justify
progress into phase I and hopefully more advanced clinical trials in cancer patients.
2. Materials and Methods
2.1. Preparation of Compound XII
Using sodium heparin as the starting material, the sulfonic acid group at position 2 ofuronic acid was removed under alkaline heating conditions to form a 2,3 epoxy structure.
The epoxy was then hydrolyzed under neutral conditions by heating to generate a vicinal
diol structure followed by oxidation to break the C–C bond of the ortho-diol structure to
generate a derivative with two aldehyde groups. Finally an oxidizing agent was added
to oxidize the aldehyde group into a carboxyl group to obtain the heparin derivative
containing the characteristic structure of tricarboxylic acid. The products were separated

Cells 2024, 13, 211

3 of 18

through column chromatography and the components of approximately 10 kDa werecollected and purified through ethanol precipitation to obtain compound XII.
2.2. Heparanase Enzymatic Activity
2.2.1. Fondaparinux (Arixtra)
The assay colorimetrically measures the appearance of the disaccharide product ofheparanase-catalyzed cleavage of the pentasaccharide fondaparinux (Arixtra) using thetetrazolium salt WST-1. Because this assay has a homogeneous substrate with a single
point of cleavage, the kinetics of the enzyme can be reliably characterized [26]. Assaysolutions (100 µL) were composed of 40 mM sodium acetate buffer (pH 5.0) and 100 mM
fondaparinux (Arixtra) with or without inhibitor [26]. Recombinant active heparanase,produced as described in [24], was added to a final concentration of 150 ng/mL to start theassay. The plates were incubated at 37 ◦ C for 4–24 h and the reaction was stopped by theaddition of 100 µL solution containing 1.69 mM 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H5-tetrazolio]-1,3-benzene disulfonate (WST-1) in 0.1 M NaOH. The plates were developed
at 60 ◦ C for 60 min, and the absorbance (OD) was measured at 584 nm [26].
2.2.2. FRET Analysis
Heparanase enzymatic activity was determined using homogeneous time-resolved
fluorescence energy transfer-based assay (TR-FRET), essentially as instructed by the manufacturer (Cisbio Bioassays S.A.S., Codolet, France) [27]. Recombinant active heparanase(GS3-linker 58 kDa) was purified and used for the assay. Test compounds were dissolved
and diluted using buffer containing 50 mM Tris-HCl (pH 7.4), 0.15 M NaCl, 0.1% proteasefree BSA, and 0.1% CHAPS. The diluted compound XII (3 µL) was mixed with 3 µL
heparanase (0.9 ng) in 0.2 M acetic acid buffer (pH 5.5) and 6 µL of assay buffer (pH 5.5) [27]to a final volume of 12 µL in a 384-well low-volume microplate. The enzyme activity was
detected by determining the HTRF signal ratio of 665/620 nm in a FLUOstar OMEGA
reader (BMG Labtech, Ortenberg, Germany). Each reaction was run in triplicate.
2.2.3. 35 Sulfate-Labeled ECM
Extracellular matrix (ECM) substrate deposited by cultured corneal endothelial cells
closely resembles the subendothelial basement membrane in its composition, biological
function, and barrier properties. Briefly, metabolically sulfate (35 S)-labeled ECM depositedby corneal endothelial cells was incubated with recombinant heparanase or cell lysate,and HS degradation fragments released into the incubation medium were analyzed bygel filtration. Detailed information about the preparation of sulfate-labeled ECM and its
use for the heparanase assay has been previously described [28–31]. The assay is highly
sensitive and suitable for measurements of heparanase activity in intact cells, extracts ofcells and tissues, plasma, and bodily fluids. It is semi-quantitative and time-consuming.
Briefly, sulfate [35 S]-labeled ECM coated on the surface of 35 mm tissue culture dishes was
incubated (4 h, 37 ◦ C, pH 6.0, 1 mL final volume) with recombinant active heparanase [24](2.0 ng/mL) in the absence and presence of the inhibitory molecule. The reaction mixture
contained 50 mM NaCl, 1 mM DTT, 1 mM CaCl2 , and 10 mM phosphate-citrate buffer,
pH 6.0. To evaluate the occurrence of proteoglycan degradation, the incubation medium
was collected and applied for gel filtration on a Sepharose 6B column (0.9 × 30 cm).
Fractions (0.2 mL) were eluted with PBS and counted for radioactivity. The excluded
volume (Vo) was marked by blue dextran and the total included volume (Vt) was marked
by phenol red. Degradation fragments of HS side chains were eluted from Sepharose 6B at
0.5 < Kav < 0.8 [29].
2.3. Cells
Human (Mia PaCa-2, BxPC-3, AsPC-1) and mouse (Panc02) pancreatic cancer cell lines,
human (MDA-MB-231) and mouse (4T1, Luc4T1) breast carcinoma cell lines, the human
MSTO-211H mesothelioma cell line, the human SiHa cervical carcinoma cell line, and theCells 2024, 13, 211

4 of 18

mouse MPC-11 myeloma/plasmacytoma cell line were purchased from the American Type
Culture Collection. Cell lines were confirmed to be pathogen-free, and human cell lines
were authenticated to confirm their origin before use. Cells were cultured in Dulbecco’s
Modified Eagle Medium (DMEM) or RPMI medium (Biological Industries, Beit Haemek,
Israel) containing 10% FBS and maintained at 37 ◦ C in a humidified incubator with 5% CO2
and 95% air. Cells were passed in culture no more than 2 months after being thawed fromauthentic stocks. Preparation of cell/tissue lysates and immunoblotting were performed
essentially as described.
2.4. Cancer Models
Mice were housed in a pathogen-free facility with access to food and water ad libitum. All animal studies were performed in compliance with the regulations and ethicalguidelines for experimental animal studies, in accordance with the Technion’s Institutional
Animal Care and Use Committee (IL-078-05-21; OPRR-A5026-01). At the time of sacrifice,tumors were removed to determine the primary tumor burden. Gross metastatic eventswere identified through visual inspection of lymph node basins, liver, peritoneum, diaphragm, lungs, and spleen. Samples were fixed in 10% formalin or snap-frozen in liquid
nitrogen for further studies.
2.4.1. Pancreatic Ductal Adenocarcinoma (PDAC)
Human (Mia PaCa-2, BxPC-3, AsPC-1, incompetent model) PDAC cells from exponential cultures were dissociated with trypsin/EDTA, washed with PBS, and brought toa concentration of 5 × 107 cells/mL. The cell suspensions (5 × 106 /0.1 mL) were subcutaneously inoculated in the right flank of 6–8-week-old SCID mice, respectively. When
palpable tumors were noticed (Days 8–10), mice were randomly assigned to cohorts (8 miceeach) receiving vehicle, compound XII (20–160 mg/kg/once a day, s.c.), or gemcitabine(30 mg/kg twice weekly or 50 mg/kg weekly). Tumor size was determined twice or thrice aweek by externally measuring tumors in two dimensions using a caliper. Tumor volume (V)was determined by the equation V = L × W2 × 0.52, where L is the length and W the widthof the xenograft. At the end of the experiment, mice were sacrificed and xenografts were
resected, weighed, and fixed in formalin for pathological examination and immunostaining.
2.4.2. PDX Model
Tumor fragments from donor mice were harvested and used for inoculation into mice,essentially as described in [32]. Each mouse was subcutaneously inoculated in the rightupper flank with primary human pancreatic PDX PA1233 tumor fragment (2–3 mm indiameter) [32]. Before dosing with drugs, all animals were weighed and tumor volumes
were measured with calipers. When the average tumor volume reached 128.0 mm3 , themice were randomized into groups according to their tumor sizes. Since the tumor volume
would affect the effectiveness of the treatment, randomization was based on tumor volume
to ensure that the tumor volumes between different groups were similar.
2.4.3. Breast Carcinoma
Luc-4T1 murine breast carcinoma cells were injected (5 × 105 cells/mouse) into thethird mammary fat pad of BALB/c mice. Two days after cell inoculation, mice weredivided into groups (n = 8 mice/group) and treated (i.p.) with vehicle (PBS), Docetaxel(10 mg/kg, i.p. QWx4), or compound XII (i.p. 20–80 mg/kg/once a day). The primarytumors were excised on Day 13 and weighed. Treatment continued as described above andthe mice were evaluated for the formation of lung metastases (Day 28—IVIS bioluminescentimaging) and survival time. MDA-MB-231 human breast carcinoma cells (5 × 106 /mouse,n = 8–10/group) were inoculated orthotopically into SCIS/NOD mice. Mastectomy was
performed on Day 13 and the mice were monitored for tumor volume and survival time.

Cells 2024, 13, 211

5 of 18

2.4.4. Mesothelioma
Luciferase-labeled MSTO-211H human mesothelioma cells were i.p. inoculated(2 × 106 /0.2 mL) into NOD/SCID mice. Ten days after cell inoculation, mice were randomly assigned to cohorts (7 mice each) receiving vehicle, compound XII (s.c. 80 mg/kg/oncea day), or cisplatin (3 mg/kg; once/3 weeks), all administered i.p. Tumor development wasinspected (once a week) by IVIS imaging following administration of luciferin [14].
2.4.5. Myeloma
MPC-11 mouse myeloma/plasmacytoma cells [33] were injected (0.5 × 106 /mouse,s.c., n = 8/group) into BALB/c mice. Mice were treated with compound XII (40 mg/kg;
once a day, starting on Day 3). Tumor volume was measured on Days 9, 11, and 14. Tumors
were excised on Day 14 and weighed.
2.4.6. IVIS Imaging
Bioluminescent imaging of luciferase-expressing tumors was performed using a highly
sensitive, cooled charge-coupled device (CCD) camera mounted in a light-tight specimenbox (IVIS; Xenogen Corp., Waltham, MA, USA). Imaging was performed in real-time, wasnon-invasive, and provided quantitative data. Briefly, mice were intraperitoneally injectedwith D-luciferin substrate at 150 mg/kg, anesthetized, and placed onto a warmed stage
inside the light-tight camera box, with continuous exposure to isoflurane (EZAnesthesia,
Palmer, PA, USA). Light emitted from bioluminescent cells was detected by the IVIS
camera system with images quantified for tumor burden using a log-scale color range set
at 5 × 104 to 1 × 107 and total photon counts per second (PPS) was measured using Living
Image (Xenogen) [14].
2.5. Western Blot Analysis
Nude mice (n = 6/group) s.c. inoculated with Mia PaCa-2 cells were treated withincreasing amounts of compound XII (daily injections of 20, 40, or 80 mg/kg) or vehicle
alone. On Day 25, the tumors were resected and the tumor tissues (50 mg) were lysed in
500 µL of RIPA buffer. After incubation on ice for 30 min, the lysates were centrifuged andsupernatants were collected. Total protein was determined using the BCA method and50 µg of protein was applied to SDS-PAGE. Western blot analysis was carried out using
anti-heparanase antibody (Proteintech 66226-1-ig, Rosemont, IL, USA) and anti-GAPDH
polyclonal antibodies.
2.6. Heparanase Uptake and Processing
Uptake experiments were carried out essentially as described in [30]. Briefly, cells(SiHa cervical carcinoma) were incubated with 65 kDa latent heparanase purified as described in [28] (1 µg/mL; serum-free conditions) in the absence and presence of increasing
concentrations (1, 5, 10 ug/mL) of compound XII. Twenty-four hours later, the medium was
aspirated, cells were washed three times with ice-cold PBS, and cell extracts were subjected
to immunoblotting with polyclonal anti-heparanase antibody (ANT-155, ProSpec-Tany,
TechnoGene Ltd., Ness-Ziona, Israel), which detects both the latent (65 kDa) and active(8 + 50 kDa) enzyme.
2.7. Pharmacokinetics
A total of 24 Sprague-Dawley rats (12 females, 12 males) were subcutaneously or
intravenously injected with a single dose of compound XII. For the intravenous injection
and subcutaneous injection, each animal was given 15 mg/kg. This dosage was basedon the calculation of surface area between mouse and rat. Selecting the lowest effective
dose (20 mg/kg in mice) was for the consideration of safety when the compound was i.v.
injected. Whole blood was collected at 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and24 h post-injection. The concentration in the plasma was measured by LCMS. The plasma
sample was processed by liquid–liquid extraction in phenol: methylene chloride (v/v, 2:1)

Cells 2024, 13, x FOR PEER REVIEW

Cells 2024, 13, 211

6 of 18(20 mg/kg in mice) was for the consideration of safety when the compound was i.v. injected. Whole blood was collected at 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h6 of 18
post-injection. The concentration in the plasma was measured by LCMS. The plasma sample was processed by liquid–liquid extraction in phenol: methylene chloride (v/v, 2:1) andand
300ofµLof supernatant
was transferredmL centrifuge
tubes
in 2.00
which
thenthen
300 µLsupernatant
was transferred
to 0.6 tomL0.6
centrifuge
tubes inwhich
µL2.00of µL
offormicacid
added.
Then,
the samples
hydrolyzed
in a waterbathformicacid
waswasadded.
Then,
the samples
werewerehydrolyzed
in a water
bath (90°C)(90for ◦atC) forleast
20 hr.
Xevo Xevo
TQ-S mass
with the electrosprayionization source
at
least
20 A
hr.Waters
A Waters
TQ-Sspectrometer
mass spectrometerwith the electrosprayionization
operatedin negative
scan modewasusedwasto monitor
precursor
product ion
transi- ionsourceoperatedin negative
scan
mode
used tothe
monitorthe toprecursor
to producttions of m/zof
390.03and 261.60
compound
XII and 5-Fluorouridine(the internal
standtransitions
m/z 390.03
andfor261.60for compound
XII and 5-Fluorouridine(theinternal
TM WinNonlin
TM WinNonlin
ard), respectively.
Phoenix
8.1 was8.1
usedcalculatethe PK theparametersofstandard),
respectively.
Phoenix
wastoused
to calculate
PK parameterscompoundXII
byemploying
non-compartmental
model
analysis.
of compound XII by employing non-compartmental model analysis.
The radioactive
radioactive 8989
Zra attached
ached to theterminal
aldehyde
groupof compound
The
Zrlabellabelwaswasto theterminal
aldehyde
groupof compound
89Zr-XII
89
XII.
The
chemicalpurityandRCP(radiochemicalpurity)
of
was
≥
90%,
measured
XII. The chemical purity and RCP (radio chemical purity) of Zr-XII was ≥90%,
measuredusing aa 1500
1500 series
series HPLC
detector.
The
relative
biologiusing
HPLC coupledcoupledwith
withananonline
onlineradiation
radiation
detector.
The
relative
biologicalcal activity(heparanase-inhibitingactivity)
activity) ofof 89
equivalent
to thatof unlabeled
89Zr-XII
activity(heparanase-inhibiting
Zr-XIIwas
wasequivalent
to thatof unlabeled
89 Zr-XII following a single subcutaneouscompound XII.
of of
89 Zr-XII following a single subcutaneouscompound
XII. The
Thetissue
tissuedistribution
distributionproﬁleprofileadministration in 4T1 tumor-bearing mice was scanned by static PET/CT for 10–30 min at
administration in 4T1 tumor-bearing mice was scanned by static PET/CT for 10–30 min ateach time point and the images were collected for analysis using the software of PMOD
each time point and the images were collected for analysis using the software of PMOD
(PMOD Technologies, Billerica, MA, USA).

(PMOD Technologies, Billerica, MA, USA).
2.8. Statistics

2.8. Statistics

Data are presented as the mean ± SEM. Statistical signiﬁcance was analyzed by 2Data are presented as the mean ± SEM. Statistical significance was analyzed bytailed Student’s t-test or one-way ANOVA. A value of p ≤ 0.05 was considered signiﬁcant.
2-tailed Student’s t-test or one-way ANOVA. A value of p ≤ 0.05 was considered signifDatasets passed the D’Agostino–Pearson normality test (Prism 5 utility software,
icant. Datasets passed the D’Agostino–Pearson normality test (Prism 5 utility software,
GraphPad, USA).

GraphPad, USA).
3. Results

3. Results
3.1. Preparation of Dicarboxylated Oxy-Heparin (DcoxyH = compound XII)
3.1. Preparation of Dicarboxylated Oxy-Heparin (DcoxyH = compound XII)Compound XII was prepared by two-step oxidation of heparin. In the ﬁrst oxidation
Compound XII was prepared by two-step oxidation of heparin. In the first oxidation
step, periodate oxidation of heparin’s nonsulfated uronic acid residues led to oxy-hepastep, periodate oxidation of heparin’s nonsulfated uronic acid residues led to oxy-heparins
rins (oxyHs), characterized by the split of the C2–C3 linkage and the formation of two(oxyHs), characterized by the split of the C2–C3 linkage and the formation of two aldehyde
aldehyde groups [25]. These were further oxidized to carboxy groups yielding dicarboxgroups[25]. These were further oxidized to carboxy groups yielding dicarboxylated oxyylated oxy-heparins (DCoxHs). Oxidation of the aldehyde to carboxyl was performed usheparins(DCoxHs).
Oxidationof the aldehyde to carboxyl was performed using sodium
ing sodium
chlorite (NaClO
2) in aqueous media, pH 4, at 0 °C for 24 h [25] (Scheme 1).
◦chlorite (NaClO2 ) in aqueous media, pH 4, at 0 C for 24 h [25] (Scheme 1).

Scheme 1.
1. Chemical
Oxy-Heparin(= DcoxyH= XII).
SchemeChemicalstructure
structureofofdicarboxylated
dicarboxylated
Oxy-Heparin(= DcoxyH= XII).

Compound XII
heparanase
enzymatic
activCompound
XII waswasexaminedexaminedfor
foritsitscapacity
capacitytotoinhibit
inhibitheparanase
enzymatic
activity
ityapplying
by applying
the Arixtra
colorimetricassay(FigureFRET
assay(Figureby
the Arixtra
colorimetricassay(Figure1A),1A),
FRET
assay(Figure1B),1B),
andandsulfatesulfate-labeled
ECM degradation(Figurepurpose,recombinantheparanase
hepalabeled
ECM degradation
assayassay(Figure1C). 1C).
For Forthisthis
purpose,recombinantranasewas incubatedwith fondaparinux(Arixtra)inabsence
the absence
presenceof increas- conwas
incubatedwith fondaparinux(Arixtra)in theandandpresenceof increasing
ing
concentrations
ofcompoundXII.
Cleavageofthepentasaccharide
substrate
a dicentrations of compound XII. Cleavage of the pentasaccharide substrate into ainto
disaccharidesaccharide
andtrisaccharide
products
wascolorimetricallyevaluated
at584
nm
following
and trisaccharide products was colorimetrically evaluated at 584 nm following the addition
theWST-1
addition
of As
WST-1
[26]. As demonstrated
in Figure
1A, compound
XII washighlyof[26].
demonstrated
in Figure 1A,
compoundXII was highly
activeinacinhibittive in inhibiting the enzyme, yielding an IC50 of 71 ng/mL. Measurements of heparanase
ing the enzyme, yielding an IC50 of 71 ng/mL. Measurements of heparanase enzymatic
activity at various concentrations of compound XII using the FRET assay [27] yielded an
6
IC50 of 241 ng/mL (Figure 1B). Compound XII was then examined for inhibition of HS
degradation using naturally produced ECM as the substrate. The results are presented as
the actual gel filtration profile of sulfate-labeled HS degradation products released into the
incubation medium (Figure 1C). Notably, compounds XII and SST0001 (= Roneparstat, aCells 2024, 13, 211

enzymatic activity at various concentrations of compound XII using the FRET assay [27]yielded an IC50 of 241 ng/mL (Figure 1B). Compound XII was then examined for inhibition of HS degradation using naturally produced ECM as the substrate. The results are 7 of 18
presented as the actual gel ﬁltration proﬁle of sulfate-labeled HS degradation products
released into the incubation medium (Figure 1C). Notably, compounds XII and SST0001(=leadRoneparstat,a lead anti-heparanasenon-anticoagulant
glycol-split
heparin)[25,34]
exanti-heparanasenon-anticoagulant
glycol-split heparin)[25,34]
exhibited
comparable
hibited comparable heparanase-inhibiting activity. We relied on the ECM assay given that
heparanase-inhibiting activity. We relied on the ECM assay given that the Arixtra assay
the Arixtra assay uses a synthetic pentasaccharide substrate, while ECM is a naturally
uses a synthetic pentasaccharide substrate, while ECM is a naturally produced substrate
produced substrate and hence be er predicts the inhibitory eﬀects of compounds in vivo.

and hence better predicts the inhibitory effects of compounds in vivo.

Figure1. 1.Heparanase-inhibiting
Recombinant heparanase
heparanase (150(150ng/mL)
FigureHeparanase-inhibitingactivity
activityofofcompoundcompoundXII.
XII. (A)(A) Recombinant
ng/mL) was incubated (18 h,◦37 °C) with fondaparinux (Arixtra) in the absence and presence of inwas incubated (18 h, 37 C) with fondaparinux (Arixtra) in the absence and presence of increascreasing concentrations (12.5–800 ng/mL) of compound XII. Cleavage of the pentasaccharide subing concentrations (12.5–800 ng/mL) of compound XII. Cleavage of the pentasaccharide substrate
strate into disaccharide and trisaccharide products was colorimetrically evaluated at 584 nm followinto
and trisaccharide
was colorimetricallyevaluated
at 584nm
followinging
thedisaccharide
addition of WST-1.
(B) Increasing products
concentrationsof compound XII wereincubated
(4 h)
with
the
additionof
WST-1.
(B)
Increasing
concentrations
ofcompoundXII
wereincubated(4
recombinant heparanase and biotin-labeled HS substrate (CisBio). Heparanase enzymatic activity h) withwasdeterminedheparanaseby the FRET andassay,
as described inHS
[28],
yielding(CisBio).
an IC50 = Heparanase
241 ng/mL. (C)Inhibition activity
recombinantbiotin-labeled
substrate
enzymatic
ofwas
ECM
degradation
by
XII.
Recombinant
heparanase(2
ng/mL)wasincubated
(4h,
37
°C)
with
determined by the FRET assay, as described in [28], yielding an IC50 = 241 ng/mL.
(C)sulInhibition
fate-labeled ECM in the absence and presence of 0.2 and 1.0 µg/mL of compound XII or SST0001 (=◦of ECM degradation by XII. Recombinant heparanase (2 ng/mL) was incubated (4 h, 37 C) with
Roneparstat). Sulfate-labeled HS degradation products released into the incubation medium were
sulfate-labeled
ECM inontheabsence 6B.
and presence of 0.2 and 1.0 µg/mL of compound XII or SST0001
subjected
to gel ﬁltration
Sepharose(= Roneparstat). Sulfate-labeled HS degradation products released into the incubation medium were
subjected
to gelfiltration on Sepharose 6B.
3.2.
Pancreatic
Canceris known to participate in the progression of PDAC, and elevated levels
3.2.Heparanase
Pancreatic Cancerof heparanase in patients with PDAC correlate with worse overall survival [35]. Taking
Heparanase
is known istocontributed
participateby
in boththe progression
PDAC,
and elevatedinto accountthat heparanasetumor cells of
andhost immune
cells, levelsofheparanaseinpatients
with
PDAC
correlate
with
worse
overallsurvival[35].
we applied mouse (Panc02) and human (MiaPaca-2, BxPC-3, AsPC-1) PDAC cells to ex-Taking
into accountheparanaseby bothtumor
cellssystem;and host
immunecells, weamine
the eﬀectthatof compound
XIIisincontributed
syngeneic (intact
host
immuneC57BL/6)
andappliedmouse(Panc02)
andhuman(MiaPaca-2,
BxPC-3,
AsPC-1)
PDAC
cells
to
examinenon-syngeneic (impaired immune system; SCID/NOD) backgrounds, respectively.
theSeveral
effect ofcompound
XII insyngeneic(intact hostimmune system;
C57BL/6)celland nonpancreaticcancermodelswere examined.
Dependingon the pancreaticsyngeneic(impairedimmunesystem;SCID/NOD)
backgrounds,
respectively.
line, treatment with compound XII (daily 20–160 mg/kg) alone yielded tumor growth incancer
were examined.
the pancreaticcell
hibitionSeveral(TGI) pancreatic
ranging from
21%models(Mia PaCa-2,
20 mg/kg, Depending
QD) to 67%on(Mia
PaCa-2,line, treatment
with compound
XII (daily
20–160mg/kg)
alone
yieldedtumor
160mg/kg,
QD). Intermediate
TGIs (about50%) wereobtainedwiththe BxPC-3
PDAC
cellgrowth
inhibition(TGI)
ranging compoundfrom 21% XII(Mia
PaCa-2,20 mg/kg,
to 67% (MiaofPaCa-2,line.
In related
experiments,(daily80 mg/kg)
yielded QD)
50% prolongation
themedian survivalof mice inoculated
orthotopicallywith
Panc02withcells.theA BxPC-3
similar 46%
160mg/kg,
QD). Intermediate
TGIs (about50%) wereobtained
PDAC cell
mediansurvival
vs.compoundcontrol untreated
mice80was
observedin mice50%
inoculated
line. In
relatedprolongation
experiments,
XII (daily
mg/kg)
yieldedprolongation(i.p.)
with
AsPC-1survival
cells andoftreatedwith compound
XII (daily with
100 mg/kg).
of themedianmice inoculated
orthotopically
Panc02 cells. A similar 46%
In human
Miaprolongation
PaCa-2 tumor-bearing
a signiﬁcant
21%
inhibitionof tumor
mediansurvival
vs. controlmice,
untreatedmice wasobservedin miceinoculated
growthwas noted
alreadyat 20treated
mg/kg ofcompound
XII, XII
increasing
to amg/kg).
comparable 45%(i.p.) with
AsPC-1
cells andwithcompound(daily 100
inhibition
when thedrug
was administered
at 40 mice,or 80 mg/kg,
and increasing
to a compaIn human
Mia
PaCa-2
tumor-bearing
a significant
21% inhibitionof tumor
rable
70%
inhibition
when
the
drug
was
administered
at
160
mg/kg
(Figure
2A).
In
this
growth was noted already at 20 mg/kg of compound XII, increasing to a comparable
se45%
ing,inhibition
compoundwhen
XII (20mg/kg)
eﬀective as gemcitabinemg/kg,
p <increasing
0.01 vs. to athedrug waswas asadministered
at 40 or 80 (50mg/kg,
andcontrol,p > 0.05 vs. Gem), while treatment with 40 mg/kg of compound XII was morecomparable 70% inhibition when the drug was administered at 160 mg/kg (Figure 2A).
eﬀective (Figure 2A). In the BxPC-3 model, treatment with compound XII (50 mg/kg) or
In this setting, compound XII (20 mg/kg) was as effective as gemcitabine (50 mg/kg,
Gem (30 mg/kg) reduced primary tumor growth by ~50% (each, p < 0.01 vs. control) (Figp < 0.01 vs. control, p > 0.05 vs. Gem), while treatment with 40 mg/kg of compoundure 2B).
XII was more effective (Figure 2A). In the BxPC-3 model, treatment with compound XII
Next, we investigated the therapeutic eﬃcacy of compound XII in the PA1233 human(50 mg/kg) or Gem (30 mg/kg) reduced primary tumor growth by ~50% (each, p < 0.01 vs.
pancreatic cancer PDX model. Brieﬂy, tumor fragments from donor mice were harvested

control) (Figure 2B).

7

Cells 2024, 13, 211

volume of mice in the untreated control group was 1543.26 mm3. The mean tumor volumeof mice treated with compound XII (80 mg/kg) alone was 981.17 mm3 (TGI = 39.68%; p <0.01) (Figure 2C). These results indicated that compound XII was well tolerated in the
subcutaneous human pancreatic cancer PA1233 PDX model. Our results demonstrated theeﬀectiveness of compound XII in a enuating PDAC progression as a single drug in both
8 of 18
immunocompetent and incompetent model systems (Figure 2).

FigurePDACtumor
tumorgrowth.
growth.
(A)
Mia
PaCa-2
human
PDAC
Figure2.2.Compound
Compound XII
XII inhibits
inhibits PDAC
(A)
Mia
PaCa-2human
PDAC
cellscells
werewere
inoculated/mouse,s.c.,s.c.,n n= =8/group)
SCID/NOD
randomized
inoculated(0.5(0.5×× 1066/mouse,
8/group)intointo
SCID/NOD
mice.mice.
Mice Mice
were wererandomized
into 6 intoalone,
gemcitabine(Gem
50 mg/kg,
i.p. QW),
or compound
XII (20, XII
40, 80,
6 groups
groups receiving
receivingvehicle
vehiclealone,
gemcitabine(Gem
50 mg/kg,
i.p. QW),
or compound(20, 40,oror
160
mg/kg,
QD,QD,s.c.).s.c.).
Tumor
volumewas measured
twice atwice
weekaasdescribed
in ‘Methods’.
(B)
80,
160
mg/kg,
Tumor
volumewas measured
week
as describedin ‘Methods’.
Compound XII inhibits BxPC-3 PDAC tumor growth. BxPC-3 human PDAC cells were inoculated(B) Compound
XII inhibits BxPC-3 PDAC tumor growth. BxPC-3 human PDAC cells were inoculated(0.5 × 1066/mouse, s.c., n = 8/group) into SCID/NOD mice. Mice were randomized into 4 groups re(0.5× 10vehicle/mouse,s.c.,gemcitabinen = 8/group)
into
mice.
Mice wereinto
4 groups
ceivingalone,(Gem
30 SCID/NOD
mg/kg, i.p. BIW),
compoundXIIrandomized(50 mg/kg, QD,s.c.),
or
receivingvehiclealone,
gemcitabine
30volume
mg/kg,wasi.p. measured
BIW), compound
XII (50
QD,
compoundXII (100mg/kg,
QD, s.c.).(GemTumorthrice a week
asmg/kg,described
in s.c.),(C)XII
Compound
XII inhibits
PDAC
tumor
growthin measured
a PDX model.
PA1233tumor
fragor‘Methods’.
compound(100 mg/kg,
QD, s.c.).
Tumor
volumewasthricea weekas described(2–3 mmdiameter) were
into BALB/c
nude
mice. PA1233
Mice wereinments‘Methods’.
(C)inCompound
XII subcutaneously
inhibits PDAC inoculated
tumor growth
in a PDX
model.
tumor
randomized(n = 8/group)
receiving vehicle
or compound
XII (80 mg/kg,
QD, s.c.).
fragments(2–3intomm2 groups
in diameter)
were subcutaneously
inoculated
into BALB/cnude mice.
Mice wererandomized into 2 groups (n = 8/group) receiving vehicle or compound XII (80 mg/kg, QD, s.c.).
3.3. Breast Cancer

Givenwetheinvestigatedinvolvementtheof heparanase
the pathogenesisof breast
cancer[36–40], humanwe
Next,therapeuticinefficacy
of compound
XII inthe PA1233
investigatedthe eﬀect
compound
XIItumoron breast
tumorigenicity.
Brieﬂy,
MDA-MB-231
pancreaticcancer
PDX ofmodel.
Briefly,fragmentsfrom donor
mice
were harvested
human
carcinomainto
cells
were BALB/c
inoculated(s.c.)
intoEach
SCIS/NOD
Mice wereandusedbreast
for inoculation
femalenude
mice.
mouse mice.
was subcutaneously
treatedwithcompoundXII
starting
on
Day
10
and
the
tumor
volumewasmeasured
twice
inoculated in the right upper flank with primary human pancreatic PDX PA1233
tumor
a
week.
As
demonstratedin
Figure3A,
signiﬁcant
inhibition(~20%)of
MDA-MB-231
fragment (2–3 mm in diameter). On Day 54 after drug administration, the meanpritumor
mary tumor
growthnoted already20 mg/kg
compoundincreasing
to a comvolumeof micein thewas
untreated
controlatgroupwasof1543.26
mm3XII,. The
mean tumor
volume58% inhibition
when the drug
wasmg/kg)
administered
40 or981.17
80 mg/kg.
of the3 (TGI
ofparable
mice treatedwith compound
XII (80
aloneatwas
mmAll= ani39.68%;
mal treatments were well tolerated. Notably, treatment with compound PG545 (= Pixp < 0.01) (Figure 2C). These results indicated that compound XII was well tolerated in theatimod, a cholestanol-sulfo-tetrasaccharide) [41], yielded a TGI of 59-77% on MDA-MBsubcutaneous human pancreatic cancer PA1233 PDX model. Our results demonstrated the
231 primary tumor growth, but this was associated with 4–11% loss in body weight.
effectiveness of compound XII in attenuating PDAC progression as a single drug in bothimmunocompetent and incompetent model systems (Figure 2).

3.3. Breast Cancer
Given the involvement of heparanase in the pathogenesis of breast cancer [36–40], we
investigated the effect of compound XII on breast tumorigenicity. Briefly, MDA-MB-231
human breast carcinoma cells were inoculated (s.c.) into SCIS/NOD mice. Mice weretreated with compound XII starting on Day 10 and the tumor volume was measured twice aweek. As demonstrated in Figure 3A, significant inhibition (~20%) of MDA-MB-231 primarytumor growth was noted already at 20 mg/kg of compound XII, increasing to a comparable
58% inhibition when the drug was administered at 40 or 80 mg/kg. All of the animaltreatments were well tolerated. Notably, treatment with compound PG545 (= Pixatimod, acholestanol-sulfo-tetrasaccharide) [41], yielded a TGI of 59-77% on MDA-MB-231 primarytumor growth, but this was associated with 4–11% loss in body weight.
Next, we examined the efficacy of compound XII in a survival model of mouse 4T18breast cancer. Briefly, 4T1 breast cancer cells were orthotopically inoculated into the third
mammary gland of BALB/c mice. Mice were then randomized into 5 groups (n = 8) treatedwith vehicle alone, compound XII (daily injections of 20, 40, or 80 mg/kg starting on
Day 1), or docetaxel (weekly, 10 mg/kg, starting on Day 10). All mice were subjected tomastectomy on Day 13. In this model, compound XII-treated animals showed prolonged
median survival compared with vehicle control (44-49 vs. 38.5 days) or Docetaxel therapy(42.5 days, p = 0.012) (Figure 3B).

Cells 2024, 13, x FOR PEER REVIEW

9 of 18

Cells 2024, 13, 211

9 of 18

Figure 3. Compound XII inhibits breast tumor growth. (A) MDA-MB-231 TNBC model. MDA-

Figure3. Compound
XII inhibits
breasttumor(A) MDA-MB-231
TNBC model.
MB-231
human breast
carcinomacells(5 × growth.
105 /mouse,s.c., n = 8–10/group)
wereMDA-MBinoculated (s.c.)
231 human breast carcinoma cells (5 × 105/mouse, s.c., n = 8–10/group) were inoculated (s.c.) into
into SCID/NOD mice. Mice were randomized into 4 groups receiving vehicle alone, compound
SCID/NOD mice. Mice were randomized into 4 groups receiving vehicle alone, compound XII (20XII (20QD,
mg/kg,
QD, s.c.), XII
compoundXIIQD,(40s.c.),mg/kg,
QD, s.c.),XIIor(80compound
XII(80Tumor
mg/kg, QD,
mg/kg,s.c.), compound(40 mg/kg,
or compound
mg/kg, QD,s.c.).
s.c.). Tumor
volume wasa week.XII(B)prolongs
Compound
XII prolongs
thecancer-bearsurvival of breast
volumewas measured
twicemeasured
a week. (B)twice
Compoundthe survivalof breast
cancer-bearing
micemousein the breast
4T1 mousecancer mastectomy
model. breast
4T1 mousebreastcells
carcinomaing
mice in the 4T1
cancerbreastmastectomy
model. 4T1 mouse
carcinoma(50,000
cells/mouse,n = 8/group)
were inoculated
into the mammary
gland of BALB/c
Mice mice.
cells (50,000
cells/mouse,n = 8/group)
were inoculated
into the mammary
glandmice.
of BALB/c
were
on Dayon
1 intoreceivingvehiclevehicle
alone, docetaxel(10 mg/kg,
i.p. QW,i.p. QW,
Micerandomized
were randomized
Day 51 groups
into 5 groups
receivingalone, docetaxel(10 mg/kg,
weekly from Day 10), compound XII (20 mg/kg, QD, s.c., daily starting on Day 1), compound XII
weekly from Day 10), compound XII (20 mg/kg, QD, s.c., daily starting on Day 1), compound XII(40 mg/kg, QD, s.c., daily starting on Day 1), or compound XII (80 mg/kg, QD, s.c., daily starting on(40 1).
mg/kg,
QD, s.c.,daily
startingDaycompound(80wasmg/kg,
QD, fors.c.,70dailyDay
Mastectomywasperformed
ononDay13 1),
andorsurvivalof theXII
mice
monitored
days.starting
onLung
Day metastasis.
1). Mastectomy
was performed
13 andsurvivalofvehicle
the mice
wasPaclitaxel
monitored for(C)
The experimentconsistedonof Day3 groups(10 miceeach):(PBS),(PTX)
alone,
orLungcompound
XII alone.
mice consisted
were injectedLuc-4T1
mouse
breastvehicle
carci- (PBS),
70 days.
(C)
metastasis.
The BALB/cexperimentof 3withgroups(10 miceeach):
noma
cells (50,000
Compound(80 mg/kg,
QD, mices.c.) wasadministeredon Day2 and mouse
Paclitaxel(PTX)cells/mouse).
alone, or compoundXIIXIIalone.
BALB/cwere
injected with
Luc-4T1
once a day thereafter. PTX (10 mg/kg) was administered on Day 9. The primary tumors were rebreast carcinoma cells (50,000 cells/mouse). Compound XII (80 mg/kg, QD, s.c.) was administeredsected on Day 14 and weighed. Treatment was continued as described above and the mice evaluatedonlungDaymetastases
2 and once(IVIS),
a day asthereafter.
(10 mg/kg)
administeredon Day9. TheprimaryfordescribedPTXin ‘Materials
andwas
Methods’.
Shown areimagestaken
on day tumors
wereresected
on
Day
14
and
weighed.
Treatment
wascontinued
asdescribed
above
and the mice28.

evaluated for lung metastases (IVIS), as described in ‘Materials and Methods’. Shown are images
Next,
we 28.
examined the eﬃcacy of compound XII in a survival model of mouse 4T1
taken
on daybreast cancer. Brieﬂy, 4T1 breast cancer cells were orthotopically inoculated into the third
3.4. Breastgland
Cancermammaryof Metastasis
BALB/c mice. Mice were then randomized into 5 groups (n = 8) treatedwith vehicle
alone, compound
XIIof(daily
injections
orbreast
80 mg/kg
startingmodel.
on DayBriefly,
We investigatedthe effectcompound
XIIof
in20,
the40,
4T1
carcinoma1),
or
docetaxel(weekly,
10
mg/kg,
starting
on
Day
10).
All
mice
were
subjected
tomasLuc-4T1 murine breast carcinoma cells were injected (50,000 cells/mouse) into
the third
tectomy
on
Day
13.
In
this
model,
compound
XII-treatedanimals
showedprolonged
memammary fat pad of BALB/c mice. Two days after cell inoculation, mice weredivideddian
comparedwith vehicle control(44-49(i.p.)vs. 38.5
days)
or Docetaxel
therapy (PTX,
intosurvival
3 groups(n = 10 mice/group)and treatedwithvehicle(PBS), Paclitaxel(42.5
days, p once
= 0.012)10 mg/kg,
on(FigureDay 9),3B).
or compound XII (80 mg/kg; once a day). The primary tumors

were excised on Day 14 and weighed. A significant ~30% (p = 0.009) reduction in primary

3.4.
BreastCancerwas
Metastasis
tumor
weightnoted in mice treated with compound XII while the inhibitory effectWe investigatedthe eﬀect Treatmentof compound
XII in the
4T1breast carcinoma
model.tumorsof PTX
was not significant.
continued
after
removalof the primary
Brieﬂy,Luc-4T1
murinebreastcarcinoma
cells
were
injected(50,000
cells/mouse)
intoand the mice were subjected to IVIS analysis on Day 28. The results (Figure 3C)therevealedthird
mammary
pad of BALB/cmice. Twodays after
inoculation,
mice were dinearlycompletefatinhibitionof metastasis
in responseto celltreatmentwith compound
XII alone.
vided
into compound3 groups (nXII= 10alone
mice/group)andtreated than(i.p.) PTX.
with vehicle(PBS),
Paclitaxel
Notably,
was more
effective
Altogether,while
compound(PTX,
10
mg/kg,
once
on
Day
9),
or
compoundXII(80
mg/kg;
once
a
day).
The
primary
XII had only a limited effect on the growth of the primary breast tumors,
it was verytumorswere
excised on Day
and weighed.
A signiﬁcant~30% (p = 0.009) reduction in
effective
in attenuating
the14
resultingspontaneous
metastases.
primary tumor weight was noted in mice treated with compound XII while the inhibitory

3.5. Mesothelioma
Mesothelioma tumors express high levels of heparanase and exhibit high sensitivity
9
to treatment with heparanase-inhibiting compounds [14], providing a strong rationaleforvealed nearly complete inhibition of metastasis in response to treatment with compound
XII alone. Notably, compound XII alone was more eﬀective than PTX. Altogether, while
compound XII had only a limited eﬀect on the growth of the primary breast tumors, it wasvery eﬀective in a enuating the resulting spontaneous metastases.
Cells 2024, 13, 211

3.5. Mesothelioma

10 of 18

Mesothelioma tumors express high levels of heparanase and exhibit high sensitivity
to treatment with heparanase-inhibiting compounds [14], providing a strong rationale for
exploring
theeﬀecteffectofofcompoundcompoundon mesothelioma
progression.
We examined
exploring the
XIIXIIon mesothelioma
progression.
We examinedthe ef- theeffectcompoundcisplatin(cis)
MSTO-211H
human
mesothelioma
tumor
fect ofofcompoundXIIXIIor orcisplatin
(cis)
on on
MSTO-211H
human
mesothelioma
tumor
pro- pro6) were
gression.
Briefly, luciferase-labeledluciferase-labeledMSTO-211H
MSTO-211H
human
mesothelioma
cells106 ) were
gression. Brieﬂy,
human
mesotheliomacells(2 ×(2
10×
intraperitoneally(i.p.)inoculated
inoculatedinto
into
NOD/SCID
mice.
cell inoculation,
intraperitoneally (i.p.)
NOD/SCID
mice.
TwoTwodaysdays
afteraftercell inoculation,
mice
weredivided
dividedintointo
3 groups= 6–7/group)and treated(i.p.)
withvehiclemice were
3 groups(n =(n
6–7/group)and treated(i.p.) withvehicle(PBS),
com-(PBS),
pound XII (80once aonceday),a or
cisplatin(3 mg/kg;
once every
weeks).3 Tumor
compound
XIImg/kg;(80 mg/kg;
day),or cisplatin(3 mg/kg;
once3 every
weeks).deTumor
velopment
wasinspected
by
IVIS
once
a
week,
startingonDay
10
post-cell
inoculation
development was inspected by IVIS once a week, starting on Day 10 post-cell inoculation(when thethe luciferase
Treatment
withcompound
XII oralonealone(when
luciferasesignal
signalisisdetected).
detected).
Treatment
withcompound
XIIcisplatin
or cisplatinyieldedacomparable
60–80%
inhibitionoftumor
growth(IVIS
signal)measuredonyielded a comparable 60–80% inhibition of tumor growth (IVIS signal) measuredDays
on Days
10, 23,23, 30,30, andactualIVIS
signals
obtainedon Days
10 andof 23 of10,
and 3636of
ofthe
theexperiment.
experiment.The
The
actualIVIS
signals
obtainedon Days
10 23
and
the
experimentarepresented
inFigure4Aandquantiﬁed
in
the
bar
graphs,revealing
the experiment are presented in Figure 4A and quantified in the bar graphs, revealing
highly significant
signiﬁcant inhibition
growthbyby
XIIXIIat alltime
points.
Cisplatin
showedhighlyinhibitionofoftumor
tumor
growthat alltime
points.
Cisplatin
showed
no
eﬀect
on
Day
10
but
was
as
eﬀectiveascompound
XII
at
later
time
points.
Next,
we weno effect on Day 10 but was as effective as compound XII at later time points. Next,
analyzed
the
eﬀect
of
the
above
treatmentsonthesurvival
of
the
mice.
As
demonstrated
analyzed the effect of the above treatments on the survival of the mice. As demonstrated inin Figure 4B (Kaplan–Meier curves), the median survival time of mice treated with comFigure
4B (Kaplan–Meier curves), the median survival time of mice treated with compound
pound XII alone was longer than that of control untreated mice (65 vs. 48 days, p = 0.0264).
XII alone was longer than that of control untreated mice (65 vs. 48 days, p = 0.0264). SigSigniﬁcantly improved survival was also noted in mice treated with cisplatin alone (menificantly improved survival was also noted in mice treated with cisplatin alone (median
dian survival = 71 days vs. 48 days for untreated mice, p = 0.017).

survival = 71 days vs. 48 days for untreated mice, p = 0.017).

Figure 4.
4. Eﬀectonon
mesothelioma
tumor
growth.
(A) Luciferase-labeled
MSTOFigure
Effectof
ofcompoundcompoundXII
XII
mesothelioma
tumor
growth.
(A) Luciferase-labeled
MSTO6) 6were intraperitoneally (i.p.) inoculated into NOD/SCID
211H
human
mesothelioma
cells
(2
×
10
211H human mesothelioma cells (2 × 10 ) were intraperitoneally (i.p.) inoculated into NOD/SCID
mice. Two days after cell inoculation, mice were divided into 3 groups (n = 6–7/group) and treatedmice. Two days after cell inoculation, mice were divided into 3 groups (n = 6–7/group) and treated(i.p.) with vehicle (PBS) alone, compound XII (80 mg/kg; once a day), or cisplatin (Cis; 3 mg/kg; once
(i.p.)
with vehicle (PBS) alone, compound XII (80 mg/kg; once a day), or cisplatin (Cis; 3 mg/kg; once
every 3 weeks). Starting on Day 10 post-cell inoculation (when luciferase signal is detected), tumor
every
3 weeks).
onby
Day
10(bioluminescentpost-cell inoculation(when
luciferase signal istumors)detected),development
wasStarting
inspected
IVIS
imagingof luciferase-expressing
oncetumor
development
wasby IVIS(bioluminescentof keptluciferase-expressing
tumors)a week. Shownareinspected
images taken
on Days
10 and 23. (B)imaging
Mice were
for an additional 80
days toonce a
study theeﬀectof images
the abovetreatments
survival
mesothelioma-bearing
mice (plo ed
as
week.
Shown
are
taken
on Dayson10theand23. (B)ofMicewere kept for an additional
80 daystoKaplan–Meiercurves,rightpanel).
study the effect of the above treatments on the survival of mesothelioma-bearing mice (plotted as
Kaplan–Meier curves, right panel).
3.6. Mouse Myeloma

3.6. Mouse Myeloma
We examined the effect of compound XII on mouse myeloma tumor growth. Briefly,
MPC-11 mouse myeloma/plasmacytoma cells were injected into BALB/c mice. The mice
10
were treated with compound XII and tumor volume was measured on Days 9, 11, and
14 (Figure 5A). Tumors were excised on Day 14 and weighed (Figure 5B). A most pronounced inhibition of tumor growth was observed. All of the animal treatments were welltolerated. The advantage of using a syngeneic mouse model vs. immunodeficient mice is theexistence of an intact, fully active immune system that plays an important role in tumor progression and likely also in the anti-tumorigenic effect of heparanase-inhibiting compounds.

Cells 2024, 13, 211

were treated with compound XII and tumor volume was measured on Days 9, 11, and 14
inhibitionof tumor
growthwasof the(Figure
animal5B).
treatments
were well tolerated.
(Figure5A). Tumors
were
excisedon observed.
Day 14 andAll
weighedA most pronounced
The
advantage
of
using
a
syngeneic
mouse
model
vs.
immunodeficient
mice is the existinhibition of tumor growth was observed. All of the animal treatments were well tolerated.
enceof an intact,
fully
active immune
systemvs.thatplays an importantroleexistin tumor proTheadvantageof using
a syngeneic
mouse model
immunodeﬁcient
mice is theenceof an intact,
fully active
system that plays aneﬀect
importantrole in tumor progression
and likely
also immune
in the anti-tumorigenicof heparanase-inhibiting
comgression
11 of 18
pounds.and likely also in the anti-tumorigenic eﬀect of heparanase-inhibiting compounds.

Figure
5. Effectof compound
XII onmouse
MPC-11mouse
myeloma
MPC-11 mouse
Figure5. Eﬀectof compound
XII on MPC-11
myelomatumor
growth.tumor
MPC-11growth.
mouse my6n/mouse,
Figure5. Eﬀect of cells
compound
XII
on
MPC-11mouse
myelomatumor
MPC-11
myeloma/plasmacytoma
werecells
injected(0.5
× 10 6/mouse,= 8/group)intongrowth.
BALB/cmice.
Micemyeloma/plasmacytoma
wereinjected(0.5
×s.c.,10s.c.,= 8/group)
into mouse
BALB/cmice.
6starting/mouse,s.c.,n=8/group)
into
BALB/cmice.
Miceeloma/plasmacytoma
cells
were
injected
(0.5
×
10
were
treatedwithcompoundXII(40
mg/kg;
once
a
day,
on
Day
3).
(A)
Tumor
volumewasMice were treated with compound XII (40 mg/kg; once a day, starting on Day 3). (A) Tumor volume
measuredon Days9, 11,
and 14, left
panel).
(B) Tumors
excisedon Dayand(rightvolume waswere treatedwithcompoundXII(40 mg/kg;
oncewerea day,
starting
on 14Day3).weighed(A) Tumorwasmeasuredon Days inhibition
9, 11, andof14,
left panel).
(B)
Tumorswere excised on Day 14 and weighedpanel),
revealinga profoundmyelomatumor
growth.
** excisedp = 0.0031.
measuredon Days9, 11, and 14, left
panel). (B)Tumorswere
on Day 14 and weighed (right(right panel), revealing a profound inhibition of myeloma tumor growth. ** p = 0.0031.
panel), revealing a profound inhibition of myeloma tumor growth. ** p = 0.0031.
3.7. Heparanase Levels in PDAC Tumors

3.7. Heparanase Levels in PDAC Tumors
demonstrated
in Figure
1, compound
3.7.AsHeparanase
Levelsin PDAC
Tumors XII was a potent inhibitor of heparanase endemonstrated
in Figure
1, compound
XIIwewasa potentwhether
inhibitorof heparanase
zymaticAs
activity.
Applying the
Mia PaCa-2
tumor model,
investigatedcomAs demonstrated
in Figure 1,
compoundXII wasa potent
inhibitorof heparanaseenenzymatictheMia protein
PaCa-2model,
we
investigatedwhetherpound
XII also activity.
affected theApplying
levels of heparanasein tumor
PDAC tumors.
For
thispurpose,zymatic
activity.
Applying
the
Mia
PaCa-2tumor
model,
we
investigatedwhether
commice(n = 6/group)
inoculated(s.c.)thewith
Mia of
PaCa-2cells wereprotein
treated inwithincreasing
compound
XII also
affectedlevelsheparanase
PDAC
tumors. For thispound of
XII also affected
the levelsof heparanasein PDACtumors.
Forthis purpose,amounts(dailyinjectionsof 20, 40,
orprotein
80
mg/kg)
vehiclealone,
as depurpose,compoundmice (n =XII
6/group)inoculated(s.c.)
with
MiaorPaCa-2
cells weretreated with(s.c.)
with
Mia
PaCa-2cells
weretreatedwithincreasing
mice
(n
=
6/group)inoculated
scribedinFigure
2A.
On
Day
25,
the
tumorswere
resected
and
the
tumor
tissuelysates
wereincreasing amounts of compound XII (daily injections of 20, 40, or 80 mg/kg) or vehicle
amounts
compoundXIIapplying(daily injectionsof 20, and40, or80 mg/kg)polyclonalor vehicleantialone, as desubjected
toof
Westernblotting,
anti-heparanaseanti-GAPDH
alone, as described in Figure 2A. On Day 25, the tumors were resected and the tumor tissue
bodies.
Asdemonstratedin
Figure6A,
treatment
withcompoundXII(20
mg/kg/day)
rescribed in Figure 2A. On Day 25, the tumors were resected and the tumor tissue lysates were
lysates
were subjected
to Western
blotting,
applying anti-heparanaseandanti-GAPDH
sultedin a marked
50% decrease
in applying
the normalized(GAPDH) amountof heparanase. polyclonalThere
subjected
to Western
blotting,
anti-heparanaseand anti-GAPDH
antipolyclonal
antibodies.
Asdemonstratedin
Figure6A,
treatment
withcompoundXII
wasno further
reduction in responseto treatment
with higherof compound
XII,
bodies.
As demonstrated
in Figure
6A, treatment
with amounts
compoundXII (20 mg/kg/day)re(20mg/kg/day)resultedinamarked50%
decreaseinthenormalized(GAPDH)amountsimilar to the corresponding effect on tumor growth presented in Figure 2A.
sulted in a marked 50% decrease in the normalized (GAPDH) amount of heparanase. Thereof heparanase. There was no further reduction in response to treatment with higher
was no further reduction in response to treatment with higher amounts of compound XII,
amounts of compound XII, similar to the corresponding effect on tumor growth presented
similar to the corresponding effect on tumor growth presented in Figure 2A.
in Figure 2A.

Figure 6. Heparanase protein expression, uptake, and processing. (A) Compound XII a enuates
heparanase levels in PDAC tumors. Nude mice (n = 6/group) s.c. inoculated with Mia PaCa-2 cells
were treated with increasing amounts of compound XII (daily injections of 20, 40, or 80 mg/kg) or
vehicle alone, as described in Figure 2. On Day 25, the tumors were resected and the tumor tissue
Figure
6. Heparanase
Heparanaseproteinblo
expression,
uptake,andprocessing.
processing.
(A)Compound
Compound
XIIattenuates
attenuatesheplysates
were
subjected to Westerning, applying
anti-heparanase(Proteintech
66226-1-ig,
RoseFigure
6.
proteinexpression,
uptake,
and
(A)
XII
mont,
IL, USA)levels
and anti-GAPDH
polyclonal
antibodies.
positive s.c.
control(HPA 220408
GS3-PaCa-2 cells
heparanasein PDAC tumors.
Nude
mice (nThe= 6/group)
inoculated
with=Mia
aranasekDa)levelsinthe
PDAC
tumors.
Nude protein
mice (nused= 6/group)s.c. activity
inoculatedwith Mia
PaCa-2 cells werelinkerwas
puriﬁedrecombinant
the (daily
FRET
assay.
detected
were58treatedwithincreasing
amounts ofcompoundinXII
injectionsof The20, 40,or 80 mg/kg) ortreated alone,with increasing
amountsof compound
injections
20, 40, or
mg/kg)
vehiclein Figure
2. On Day XII
25, (daily
the tumors
wereofresected
and80thetumorortissuevehicleas describedalone, asdescribed
in Figure
2. Onblotting,Day 25, applying
the tumors
were resected andthe tumor66226-1-ig,
tissuelysateswerelysates
were
subjected
to Westernanti-heparanase(Proteintech
Rose11subjected
to Western
blotting, applying
anti-heparanase(Proteintech
Rosemont,
USA)
mont,
IL, USA)and anti-GAPDH
polyclonal
antibodies. The
positive 66226-1-ig,
control (HPA
220408 =IL,
GS3activity
The detected
linker
58 kDa) was the
purifiedantibodies.
recombinantThe
proteinused control
in the FRET
and anti-GAPDH
polyclonal
positive(HPA
220408assay.
= GS3-linker
58 kDa)was the purified recombinant protein used in the FRET activity assay. The detected endogenous
heparanase was presumably the 50 kDa subunit. The molecular weight of standard protein (ladder)11
was indicated. Treatment with compound XII (20 mg/kg/day) resulted in a marked 50% decreasein the normalized (GAPDH) amount of heparanase. There were no statistical differences between
the different doses of compound XII. (B) Compound XII inhibits cellular uptake and processingof exogenously added heparanase. Cervical carcinoma cells (SiHa) were left untreated (Con) or
were incubated with latent 65 kDa heparanase (1 ug/mL) together with increasing concentrations(1, 5, 10 ug/mL) of compound XII. After 24 h, cultures were washed, and cell lysate samples were
subjected to immunoblotting, applying anti-heparanase (upper panel = 50 kDa heparanase subunit)
and anti-actin (lower panel) antibodies. The polyclonal anti-heparanase antibodies (ANT-155) werefrom ProSpec-Tany, TechnoGene Ltd., Ness-Ziona, Israel).

Cells 2024, 13, 211

50% decrease in the normalized (GAPDH) amount of heparanase. There were no statistical diﬀerences between the diﬀerent doses of compound XII. (B) Compound XII inhibits cellular uptake andprocessing of exogenously added heparanase. Cervical carcinoma cells (SiHa) were left untreated(Con) or were incubated with latent 65 kDa heparanase (1 ug/mL) together with increasing concentrations (1, 5, 10 ug/mL) of compound XII. After 24 h, cultures were washed, and cell lysate samples
were subjected to immunoblo ing, applying anti-heparanase (upper panel = 50 kDa heparanasesubunit) and anti-actin (lower panel) antibodies. The polyclonal anti-heparanase antibodies (ANT155) were from ProSpec-Tany, TechnoGene Ltd., Ness-Ziona, Israel).

12 of 18

Heparanase
Uptakeand Processing
3.8.3.8.
Heparanase
Uptakeand Processing
previously
demonstrated
that secreted
or exogenously
added65latent
65 kDa hepWe We
previously
demonstrated
that secreted
or exogenously
added latent
kDa heparanaserapidlyinteracts
heparansulfateoncellthesurface,cell surface,followedby internalaranaserapidlyinteracts
withwith
heparansulfate(HS)(HS)on thefollowedby interization andand processing
processing into
into aa highly
highly active
active 50
50 ++ 88 kDa
kDa enzyme,
enzyme,aa processprocessthat
thatisis used
used by cells
nalization
attenuate
the extracellularaccumulationof heparanase[30].
We
examined
by to
cells
to a enuate
the extracellularaccumulationof heparanase[30].
We
examinedthe
theability ofcompound
XII to attenuate
the uptake
and processingof exogenously
addedrecombinantabilityof compound
XII to a enuate
the uptake
and processingof exogenously
addedrecombinantlatent heparanase.
this purpose,
cervicalcarcinoma(SiHa)
werelatent heparanase.
For thisFor
purpose,cervicalcarcinomacellscells(SiHa)
wereleftleftuntreated
untreated(Con)
were incubatedwith latent
65 heparanasekDa heparanase(1 ug/mL)
togetherwith(Con) orwereorincubatedwith latent
65 kDa(1 ug/mL)
togetherwithincreasing
increasing
concentrations
5, 10 ug/mL)of compound
XII. After
24 h, cultures
wereconcentrations(1, 5, 10(1,ug/mL)of compound
XII. After
24 h, cultures
were washed,andwashed,andcelllysatesamples
were
subjected
toimmunoblo
ing,
applying
anti-hepacell lysate samples were subjected to immunoblotting, applying anti-heparanase (upper
ranase(upperand anti-actin(lowerpanel) antibodies.
As demonstrated
in Figurepanel)
andpanel)anti-actin(lower panel)
antibodies.
As demonstrated
in Figure
6B, complete
6B,inhibition
complete inhibitionofheparanase
cellularuptakeandprocessing,reﬂectedbytheheparanase50of heparanase cellular uptake and processing, reflected by the 50 kDa
kDasubunit,
heparanasesubunit,wasobtainedinthepresence
of
10
ug/mL
of
compoundXII.
was obtained in the presence of 10 ug/mL of compound XII. Shorter incubation
Shorter
times
30–60min)towere
neededdetectboth the unprocessed
timesincubation(i.e., 30–60
min)(i.e.,
wereneededdetectbothtothe
unprocessed
65 kDa and 65
processed
kDa50andprocessed
50
kDa
forms
of
the
internalized
enzyme[30].
kDa forms of the internalized enzyme [30].
3.9.3.9.
Eﬀect
of Compound
XII onCoagulation
Parameters
Effectof Compound
XII
on Coagulation
Parameters

Compound
XII hadvery very
low anticoagulant
activity,
with about
USP 20
unit/mg
antiCompound
XII hadlow anticoagulant
activity,
with20about
USP unit/mg
antiIIa IIa
activityandnoanti-Xa
activity.
Toevaluate
the
eﬀect
in
vivo,
6
male
and
6
femaleactivity and no anti-Xa activity. To evaluate the effect in vivo, 6 male and 6 female
Sprague-Dawley rats were subjected to daily repeat-dose subcutaneous injections for 4
Sprague-Dawley rats were subjected to daily repeat-dose subcutaneous injections forweeks. Measurement of coagulation parameters on Day 29 (after last injection) showed a
4 weeks. Measurement of coagulation parameters on Day 29 (after last injection) showed
dose-dependent prolongation of aPTT (Figure 7), although the animals did not show oba dose-dependent prolongation of aPTT (Figure 7), although the animals did not show
vious bleeding. On Day 57, the aPTT value essentially returned to normal, indicating no
obvious bleeding. On Day 57, the aPTT value essentially returned to normal, indicating noaccumulation of the compound. The level of ﬁbronectin (FIB) was marginally increased
accumulation of the compound. The level of fibronectin (FIB) was marginally increased
with increased dosage, which may have been a protective feedback for the increased aPTT.
withincreaseddosage,which
may havebeen
a protective
feedback
for theincreased
Regardless,the level
returned
to normal
after theendof treatment.
Prothrombin
time(PT) aPTT.
Regardless,
the
level
returnedtonormal
after
the
end
of
treatment.
Prothrombin
was not aﬀected. The same behavior was observed with male (Figure 7, left) and femaletime (PT)was 7,
notaffected.
(Figureright)
rats. The same behavior was observed with male (Figure 7, left) and female(Figure 7, right) rats.

Figure7. Compound
XII increases
at a treatment
short treatment
but becomeswith
Figure7. Compound
XII increases
aPTT aPTT
at a shortperiod period
but becomestoleratedtolerated
with
prolonged
treatment.
Male(left)
andfemale(right)
SD
rats
were
untreated(0)ortreatedwithinprolonged treatment. Male (left) and female (right) SD rats were untreated (0) or treated withcreasing
amounts(60, 120,mg/kg/day)of compound
XII. Artery
samplescollected
increasing
amounts(60,180120,
180 mg/kg/day)of compound
XII.blood
Arteryblood were
samples
were collected
into vacuum tubes containing sodium citrate on Day 15. The blood samples were centrifuged at 1 ×
into vacuum tubes containing sodium citrate on Day 15. The blood samples were centrifuged at
800× g for 15 min at 4 °C to obtain
serum samples. Each serum sample was transferred directly into
1 ×tube800to× recordg for 15themin
4 ◦C
to obtain
samples.
seruma sample
wasExpress
transferreda testcloatingtime(APTT,serumPT) and
FIB (g/liEacher) usingStago Emo
Co- directly
into
a
test
tube
to
recordtheclottingtime(APTT,
PT)
andFIB(g/litter)
using
a
Stago
Emo
Express
agulation Analyzer.
Coagulation Analyzer.

3.10. Pharmacokinetics of Compound XII

12For evaluation of pharmacokinetics, the compound was injected (s.c. or i.v.) into
Sprague-Dawley rats, as described in ‘Materials & Methods’. Whole blood was collected at
the time points indicated in Figure 8A. The plasma concentration was measured by LCMS.
After a single intravenous injection of 15 mg/kg of compound XII, the mean value ofdrug exposure (AUClast) of compound XII in Sprague-Dawley rats was 56,951 hr*ng/mL,with a half-life (t1/2) of 0.497 hr and clearance (CL) of 4.42 mL/min/kg, which was
about 12.0% of renal blood flow (renal blood flow for rats is 36.8 mL/min/kg [42]). The
apparent volume of distribution (Vss) was 0.138 L/kg, which was about 0.207 times theCells 2024, 13, 211

was 30,067 ng/mL. The PK parameters showed no signiﬁcant diﬀerence between gender(Figure 8A).
To examine the distribution, 89Zr-labeled compound XII was injected into mice bearing 4T1 breast cancer tumors. Following single subcutaneous administration, the animals
were subjected to PET/CT scans at the indicated time points (Figure 8B). Determination of 13 of 18
tradioactivity showed distinct distribution of the compound in organs, mainly in the kidney, bone/joint, and liver. The radioactivity peak in the bladder at 2 h post-injection indicatedcompoundwas rapidly
ﬁlteredthe kidney.
TheL/kg
radioactivity
was a single
rat that
totalthebody
fluid volume(rat total
bodythroughfluid volume
is 0.668[42]). After
gradually
reduced,
but
it
still
accumulatedinthekidneyup
to72h.
Notably,theradioacsubcutaneous administration of 15 mg/kg of compound XII, the AUClast in plasma wastivitywashr*ng/mL,
elevated withtimein theofbone
joint
up to 72 h post-injection.
Nevertheless,
75,695witha t1/2
1.48andh andbioavailabilityof 139%. The
Tmax was 1 h andradioactivity
wasconstantly
detected
in
the
tumor
tissueeven
72hpost-injection,
albeitbetween
Cmax was 30,067 ng/mL. The PK parameters showed no significant differenceat agender
relatively
lower
level.
(Figure 8A).

Figure8. 8.
Pharmacokinetics
of ofCompoundXII.
(A)(A)
Naïve
rats
FigurePharmacokinetics
Compound
XII.
NaïveSprague-Dawley
Sprague-Dawleyrats(12(12females,
females,1212 males)
males)
subcutaneously
mg/kg)ororintravenously
intravenously (15(15 mg/kg)
witha single
doseof of comwerewere
subcutaneously(15(15mg/kg)
mg/kg)injected
injected
witha singledose
compound XII. Whole blood was collected at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, andpound XII. Whole blood was collected at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and24 h post-dose. The concentration in plasma was determined using LCMS (Waters Xevo TQ-S, USA).
24 h post-dose.
The8.1concentrationin plasmadeterminedofusing
LCMSXII(Waters
XevononTQ-S, USA).
TM WinNonlin
Phoenix
was used to calculate
thewasPK parameterscompoundemploying
TM WinNonlin 8.1 was used to calculate the PK parameters of compound5 XII employing
Phoenix
compartmental model analysis. (B) Mice were inoculated with 4T1 tumor cells (5 × 10 to 5 ×
89Zr-labeledmodel
analysis.
(B) Micewere
inoculated
with
4T1 tumor
cells (5 × 105 to
106non-compartmental/mouse/50 µL) under thefat padsof the thirdpair ofnippleson the left.
compound
89 Zr-labeled
XII5(approximately
100 µL)
mg/kg
and the200fatµCi/
mouse)
was
subcutaneously
injected
into
micewith a× 106 /mouse/50
under
pads
of thethird
pair of nipples
on theleft.
com3 and the animals were scanned by static PET/CT for 10–30 min at 2, 12,
tumor
volumeof≥150
mmpound XII (approximately 100 mg/kg and 200 µCi/ mouse) was subcutaneously injected into mice24, 48, and 72 h, respectively (left panel).3 At the end of each scanning, the images were reconstructedwith a tumor volume of ≥ 150 mm and the animals were scanned by static PET/CT for 10–30 min at
and analyzed using PMOD software for the delineated organs indicated. The percentage of injected
2, 12, 24, dose48, andh, respectively(left panel).
the endeach scanning,
the toimageswere reconradioactive
per72
gram
of tissue (%ID/g)
in eachAt
organ
wasof
calculated
accordingthe dose
structed
andanalyzedusing
PMOD
softwareforthedelineatedorgansindicated.
The
percentageofadministered (right panel).

injected radioactive dose per gram of tissue (%ID/g) in each organ was calculated according to thedose administered (right panel).
13

To examine the distribution, 89 Zr-labeled compound XII was injected into mice bearing
4T1 breast cancer tumors. Following single subcutaneous administration, the animals
were subjected to PET/CT scans at the indicated time points (Figure 8B). Determination of
tradioactivity showed distinct distribution of the compound in organs, mainly in the kidney,bone/joint, and liver. The radioactivity peak in the bladder at 2 h post-injection indicatedthat the compound was rapidly filtered through the kidney. The radioactivity was gradually
reduced, but it still accumulated in the kidney up to 72 h. Notably, the radioactivity waselevated with time in the bone and joint up to 72 h post-injection. Nevertheless, radioactivity
was constantly detected in the tumor tissue even 72 h post-injection, albeit at a relatively
lower level.
4. Discussion
For the most highly aggressive human cancers, such as mesothelioma, glioma, andpancreatic adenocarcinoma, durable remission is difficult to achieve and cure remains
elusive. This underscores the need to identify new therapeutic targets and strategies forhitting those targets.
Immunohistochemistry, in situ hybridization, PCR, and Western blot analysis havedemonstrated that heparanase expression is enhanced in almost all cancers examined toCells 2024, 13, 211

14 of 18

date, including, for example, myeloma, mesothelioma, breast, ovarian, pancreatic, gastric,colon, bladder, brain, prostate, and liver cancers [5,6]. Numerous clinical association studies
have consistently demonstrated that upregulated heparanase expression correlates withincreased tumor size, tumor progression, enhanced metastasis, and poor prognosis [5,6].
Notably, there is only a single, enzymatically active form of heparanase in humans, it isexpressed in very low levels in most normal tissues, and heparanase knock-out animals
exhibit no obvious deficits [43]. These characteristics imply that inhibition of heparanase
will cause minimal side effects in cancer patients. It should be admitted, however, thatanti-heparanase-based therapy has not yet been implemented in a clinical setting. Notably,
all anti-heparanase compounds that were and are being examined in clinical trials areheparin-like saccharides. While these compounds were very successful in numerous mousemodels, side effects (i.e., bleeding complications, body weight loss) and poor pharmacokinetics halted their clinical advancement in humans. Development of more specificoligosaccharides [44], small molecules [22] and neutralizing monoclonal antibodies is ongoing. Roneparstat (= SST0001), a chemically modified 100% N-desulphated, N-reacetylated,and 25% glycol-split heparin, showed a promising anti-cancerous effect in murine modelsof myeloma and sarcoma, either alone or in combination with other drugs [25], yet despite
being well tolerated, there was insignificant benefit in phase I clinical trials [18].
The current study focuses on developing a new generation of heparin derivatives
to increase the affinity to the heparanase protein. For this purpose, dicarboxylated oxyheparins (DCoxHs) containing three carboxylate groups per split residue (DC-Hep) wereprepared [25] and examined for their heparanase-inhibiting activity and effect on tumor
growth and metastasis in mouse models. After screening a series of DC-Hep compoundswith regard to the degree of deacetylation and carboxylation, molecular size, and total
charge (beyond the scope of this manuscript), the lead compound (XII) was selected
for the studies presented herein. The new compound had a similar safety profile as
Roneparstat regarding coagulation parameters, yet showed better pharmacokinetics. It isof interest to elucidate its preferential accumulation in the bone/joint. Albeit to a relatively
lower level, the compound reached breast tumor tissue, maintaining the same level up
to 72 h. Pharmacological studies showed that our newly generated lead compound XII(Scheme 1) was well tolerated and highly effective in several cancer models, including
models in which Roneparstat exhibited only a small inhibitory effect (i.e., mesothelioma,
pancreatic carcinoma). The superiority of compound XII is attributed in part to its increased
negative charge, molecular flexibility, improved affinity toward heparanase, and better
pharmacokinetics contributed by the carboxylated groups.
Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent form of pancreaticcancer, comprising 85% of cases, is very difficult to treat, and has a 5-year survival rate ofless than 5% [45]. PDAC is characterized by rapid tumor growth, late presentation, earlymetastasis, and significant resistance to conventional treatments [45]. Surgical resection
provides a potential cure; however, 80% of PDAC cases are unresectable at the time ofdiagnosis, and the majority of resected patients succumb to recurrent disease [45]. Despite
negative margins after potentially curative resection, patients who express high levels of
heparanase in their resected tumors have worse postoperative survival, suggesting that
heparanase expression confers a more aggressive tumor phenotype [35]. The efficacy ofcompound XII was evaluated against a range of PDAC cell lines, including xenograft(immunocompromised mice), syngeneic models, and a PDX model. Notably, in the Mia
PaCa-2 and BxPC-3 models, treatment with compound XII was either as effective or evenmore effective than that with gemcitabine alone (Figure 2A). Unlike the favorable effect ofcompound XII in the human PA1233 PDX model, compound XII was not effective in thePA1644 model. Notably, PA1233 is characterized by P53R282W mutation and KRASG12D
mutation, while PA1644 is P53 wild-type and KRAS wild-type, suggesting that the antitumor effect of compound XII may be related to the P53 and KRAS characteristics ofthe tumors.

Cells 2024, 13, 211

15 of 18

In women, breast cancer (BC) is the most commonly diagnosed cancer and the leading
cause of cancer death [46]. While major advances have been made toward breast cancer
prevention and treatment, the incidence of breast cancer is still increasing globally, encouraging the development of new technologies for early and more accurate diagnosis. Women
under the age of 45 account for 11% of all BC diagnoses in the United States and 9% in the
United Kingdom [47]. Moreover, a 16% increase in the incidence of BC in women aged
25–49 years is noted since the 1990s [47]. Compound XII was highly effective in models ofbreast cancer, both syngeneic and xenograft. It showed activity in the MDA-MB-231 triple
negative model, effectively inhibiting the growth of s.c. implanted tumors versus untreatedcontrols (50% inhibition at 40 mg/kg, QD) (Figure 3A). Demonstrating efficacy in a triple
negative model has positive implications for the clinical use of compound XII given that
this population of breast cancer patients is in need of treatment options because they do notrespond to HER2 inhibitors or hormonal treatments [48]. In addition to the triple negative
xenograft model, compound XII was highly effective in the 4T1 syngeneic orthotopic model.
Notably, despite a limited effect on the primary tumor, compound XII exerted a profound,nearly complete inhibition of spontaneous metastasis following resection (mastectomy) of
the primary 4T1 orthotopic breast tumor (Figure 3C). Importantly, compound XII significantly increased the survival of mice compared to the untreated group (Figure 3B). Thisis important given that metastases, appearing at various times after surgical removal of
the primary tumor, are the main cause of failure in the treatment of breast cancer. Due
to variability and extended periods, companies focus on drugs that inhibit the growth ofthe primary tumor and tend to avoid clinical trials aimed at combating metastasis. The
anti-metastatic effect of compound XII favors treatment during and right after surgicalremoval of the primary tumor.
Malignant pleural mesothelioma (MPM), the most common form of mesothelioma, is ahighly aggressive tumor characterized by diffuse local growth in the thoracic cavity [49]. It
has a poor prognosis because of difficulties in early diagnosis and resistance to conventional
therapies [50,51]. Heparanase appears to orchestrate tumor microenvironment crosstalkthat drives mesothelioma tumor progression and poor patient outcomes. Impressive
results were obtained in a model of human mesothelioma where compound XII was
effective as a single agent and nearly as potent as cisplatin (Figure 4). This result is verysignificant and encouraging given the lack of effective treatments against this rare disease.
We have previously demonstrated a profound effect of PG545 in mesothelioma models,
further supporting the involvement of heparanase in the pathogenesis of mesothelioma [14].
PG545 (= Pixatimod = cholestanol-sulfo-tetrasaccharide) has been demonstrated to possess
potent anti-tumor activity in multiple preclinical models of cancer [41]. Mice treated with
PG545 experienced body weight loss, thus limiting the maximal tolerated dose to around
20 mg/kg/week, much lower than the dose of compound XII (160 mg/kg/day) appliedin the present study. Interestingly, unlike its potent attenuation of myeloma and sarcoma
tumor growth [21], Roneparstat (100% N-desulphated, N-reacetylated, and 25% glycol-split
heparin) had little or no effect in the mesothelioma model and was less effective thancompound XII in the PDAC and breast carcinoma models. Ongoing mode-of-action studies
are aimed at elucidating the reason for these differences in potency.
We previously demonstrated that secretion of latent heparanase followed by its cellular re-uptake is a prerequisite for the delivery of latent heparanase to late endosomes
and lysosomes and its subsequent proteolytic processing and activation by lysosomal
cathepsin L. This consistent behavior indicates that heparanase is secreted but does notnormally accumulate extracellularly unless cell membrane HS is removed or competes
with heparin/heparin-like compounds [30]. Our results indicate that compound XII not
only inhibits heparanase enzymatic activity (Figure 1) but also decreases the intratumoral
levels of heparanase protein (Figure 6A) and inhibits the uptake and activation of thelatent enzyme (Figure 6B), thus providing additional mechanistic aspects for its potent
anti-tumorigenic mode of action. Given the likely ability of compound XII to interact
with heparin/HS-binding proteins other than heparanase (i.e., FGF, VEGF, HGF, HB-EGF),

Cells 2024, 13, 211

16 of 18

one cannot ascribe the observed anti-tumorigenic effects of compound XII solely to its
heparanase-inhibiting capacity.
5. Conclusions
Our present results provide a strong rationale for a phase I clinical trial aimed at testing
the safety and maximal tolerated dose of compound XII in cancer (i.e., mesothelioma,
myeloma, breast carcinoma, pancreatic carcinoma).
6. Limitations
Given that isogenic mice have limited genetic diversity, they do not represent thegenetic heterogeneity found in human cancers. Moreover, the tumor microenvironment,
immune system, and metabolism in mice differ significantly from those in humans, together
impacting how a tumor responds to a given drug.
Author Contributions: All authors participated the design of the project. U.B., N.I., M.F. and X.Z.
performed the experiments; L.L. supervised the study. I.V. and J.-P.L. wrote the paper. All authors
have read and agreed to the published version of the manuscript.
Funding: These studies were generously supported by research grants awarded to I.V. by the Israel
Science Foundation (ISF-102119), the US-Israel Binational Science Foundation (BSF-2021059), andthe Israel Cancer Association (ICA), and grants awarded to J.-P. Li by the Swedish Cancer Research
Foundation (CAN211417Pj) and the Swedish Research Council (2021-01094). The project is also
supported by the Shenzhen Science and Technology Program (JSGG20210802154545033).
Institutional Review Board Statement: The animal study protocol was approved by the Institutional
Review Board of the Technion’s Institutional Animal Care and Use Committee (IL-078-05-21; OPRRA5026-01) for studies involving animals.
Data Availability Statement: Data are contained within the article.
Acknowledgments: The support of the Shenzhen Hepalink Pharmaceutical Group and the continuous
guidance, encouragement, and assistance of the entire Hepalink team, in particular Lige Ren, Xuewen
Jin, and Baoming Huang, are greatly appreciated.
Conflicts of Interest: Author Li Li was employed by the Shenzhen Hepalink Pharmaceutical Group.
The remaining authors declare that the research was conducted in the absence of any commercial or
financial relationships that could be construed as potential conflicts of interest.

References
1.
2.
3.
4.
5.
6.
7.
8.
9.

10.

Gaskin, S.M.; Soares Da Costa, T.P.; Hulett, M.D. Heparanase: Cloning, Function and Regulation. Adv. Exp. Med. Biol. 2020,
1221, 189–229. [CrossRef]
Ilan, N.; Bhattacharya, U.; Barash, U.; Boyango, I.; Yanku, Y.; Gross-Cohen, M.; Vlodavsky, I. Heparanase-The message comes indifferent flavors. Adv. Exp. Med. Biol. 2020, 1221, 253–283. [CrossRef]
Sanderson, R.D.; Bandari, S.K.; Vlodavsky, I. Proteases and glycosidases on the surface of exosomes: Newly discovered
mechanisms for extracellular remodeling. Matrix Biol. 2019, 75–76, 160–169. [CrossRef]
Agelidis, A.; Suryawanshi, R.K.; Patil, C.D.; Campeau, A.; Gonzalez, D.J.; Shukla, D. Dissociation of DNA damage sensing byendoglycosidase HPSE. iScience 2021, 24, 102242. [CrossRef] [PubMed]
Jayatilleke, K.M.; Hulett, M.D. Heparanase and the hallmarks of cancer. J. Transl. Med. 2020, 18, 453. [CrossRef] [PubMed]
Mayfosh, A.J.; Nguyen, T.K.; Hulett, M.D. The Heparanase Regulatory Network in Health and Disease. Int. J. Mol. Sci. 2021,
22, 1096. [CrossRef] [PubMed]
Khanna, M.; Parish, C.R. Heparanase: Historical Aspects and Future Perspectives. Adv. Exp. Med. Biol. 2020, 1221, 71–96.
[CrossRef] [PubMed]
Rivara, S.; Milazzo, F.M.; Giannini, G. Heparanase: A rainbow pharmacological target associated to multiple pathologies
including rare diseases. Future Med. Chem. 2016, 8, 647–680. [CrossRef]
Purushothaman, A.; Hurst, D.R.; Pisano, C.; Mizumoto, S.; Sugahara, K.; Sanderson, R.D. Heparanase-mediated loss of nuclear
syndecan-1 enhances histone acetyltransferase (HAT) activity to promote expression of genes that drive an aggressive tumor
phenotype. J. Biol. Chem. 2011, 286, 30377–30383. [CrossRef] [PubMed]
Vlodavsky, I.; Singh, P.; Boyango, I.; Gutter-Kapon, L.; Elkin, M.; Sanderson, R.D.; Ilan, N. Heparanase: From basic research totherapeutic applications in cancer and inflammation. Drug Resist. Updates 2016, 29, 54–75. [CrossRef]

Cells 2024, 13, 211

11.
12.
13.
14.

15.

16.

17.

18.

19.

20.
21.
22.
23.
24.

25.
26.
27.
28.

29.

30.

31.

32.
33.

17 of 18

Arvatz, G.; Shafat, I.; Levy-Adam, F.; Ilan, N.; Vlodavsky, I. The heparanase system and tumor metastasis: Is heparanase the seed
and soil? Cancer Metastasis Rev. 2011, 30, 253–268. [CrossRef]
Ramani, V.C.; Zhan, F.; He, J.; Barbieri, P.; Noseda, A.; Tricot, G.; Sanderson, R.D. Targeting heparanase overcomes chemoresistanceand diminishes relapse in myeloma. Oncotarget 2016, 7, 1598–1607. [CrossRef] [PubMed]
Shteingauz, A.; Boyango, I.; Naroditsky, I.; Hammond, E.; Gruber, M.; Doweck, I.; Ilan, N.; Vlodavsky, I. Heparanase enhances
tumor growth and chemoresistance by promoting autophagy. Cancer Res. 2015, 75, 3946–3957. [CrossRef] [PubMed]
Barash, U.; Lapidot, M.; Zohar, Y.; Loomis, C.; Moreira, A.; Feld, S.; Goparaju, C.; Yang, H.; Hammond, E.; Zhang, G.; et al.
Involvement of Heparanase in the Pathogenesis of Mesothelioma: Basic Aspects and Clinical Applications. J. Natl. Cancer Inst.
2018, 110, 1102–1114. [CrossRef] [PubMed]
Takaoka, M.; Naomoto, Y.; Ohkawa, T.; Uetsuka, H.; Shirakawa, Y.; Uno, F.; Fujiwara, T.; Gunduz, M.; Nagatsuka, H.;
Nakajima, M.; et al. Heparanase expression correlates with invasion and poor prognosis in gastric cancers. Lab. Investig.
2003, 83, 613–622. [CrossRef] [PubMed]
Kundu, S.; Xiong, A.; Spyrou, A.; Wicher, G.; Marinescu, V.D.; Edqvist, P.D.; Zhang, L.; Essand, M.; Dimberg, A.; Smits, A.; et al.
Heparanase promotes glioma progression and is inversely correlated with patient survival. Mol. Cancer Res. 2016, 14, 1243–1253.
[CrossRef] [PubMed]
Sun, X.; Zhang, G.; Nian, J.; Yu, M.; Chen, S.; Zhang, Y.; Yang, G.; Yang, L.; Cheng, P.; Yan, C.; et al. Elevated heparanase
expression is associated with poor prognosis in breast cancer: A study based on systematic review and TCGA data. Oncotarget
2017, 27, 43521–43535. [CrossRef]
Galli, M.; Chatterjee, M.; Grasso, M.; Specchia, G.; Magen, H.; Einsele, H.; Celeghini, I.; Barbieri, P.; Paoletti, D.; Pace, S.; et al.
Phase I study of the heparanase inhibitor roneparstat: An innovative approach for ultiple myeloma therapy. Haematologica 2018,
103, e469–e472. [CrossRef]
Lemech, C.; Dredge, K.; Bampton, D.; Hammond, E.; Clouston, A.; Waterhouse, N.J.; Stanley, A.C.; Leveque-El Mouttie, L.;
Chojnowski, G.M.; Haydon, A.; et al. Phase Ib open-label, multicenter study of pixatimod, an activator of TLR9, in combination
with nivolumab in subjects with microsatellite-stable metastatic colorectal cancer, metastatic pancreatic ductal adenocarcinoma
and other solid tumors. J. Immunother. Cancer 2023, 11, e006136. [CrossRef]
Liu, C.J.; Chang, J.; Lee, P.H.; Lin, D.Y.; Wu, C.C.; Jeng, L.B.; Lin, Y.J.; Mok, K.T.; Lee, W.C.; Yeh, H.Z.; et al. Adjuvant heparanase
inhibitor PI-88 therapy for hepatocellular carcinoma recurrence. World J. Gastroenterol. 2014, 20, 11384–11393. [CrossRef]
Noseda, A.; Barbieri, P. Roneparstat: Development, Preclinical and Clinical Studies. Adv. Exp. Med. Biol. 2020, 1221, 523–538.
[CrossRef]
Giannini, G.; Battistuzzi, G.; Rivara, S. The Control of Heparanase Through the Use of Small Molecules. Adv. Exp. Med. Biol. 2020,
1221, 567–603. [CrossRef]
Wu, L.; Viola, C.M.; Brzozowski, A.M.; Davies, G.J. Structural characterization of human heparanase reveals insights into substrate
recognition. Nat. Struct. Mol. Biol. 2015, 22, 1016–1022. [CrossRef] [PubMed]
Naggi, A.; Casu, B.; Perez, M.; Torri, G.; Cassinelli, G.; Penco, S.; Pisano, C.; Giannini, G.; Ishai-Michaeli, R.; Vlodavsky, I.
Modulation of the heparanase-inhibiting activity of heparin through selective desulfation, graded N-acetylation, and glycol
splitting. J. Biol. Chem. 2005, 280, 12103–12113. [CrossRef]
Cassinelli, G.; Torri, G.; Naggi, A. Non-Anticoagulant Heparins as Heparanase Inhibitors. Adv. Exp. Med. Biol. 2020, 1221, 493–522.
[CrossRef]
Hammond, E.; Li, C.P.; Ferro, V. Development of a colorimetric assay for heparanase activity suitable for kinetic analysis andinhibitor screening. Anal. Biochem. 2011, 396, 112–116. [CrossRef] [PubMed]
Enomoto, K.; Okamoto, H.; Numata, Y.; Takemoto, H. A simple and rapid assay for heparanase activity using homogeneoustime-resolved fluorescence. J. Pharm. Biomed. Anal. 2006, 41, 912–917. [CrossRef]
Blich, M.; Golan, A.; Arvatz, G.; Sebbag, A.; Shafat, I.; Sabo, E.; Cohen-Kaplan, V.; Petcherski, S.; Avniel-Polak, S.; Eitan, A.; et al.
Macrophage activation by heparanase is mediated by TLR-2 and TLR-4 and associates with plaque progression. Arter. Thromb.
Vascular Biol. 2013, 33, e56–e65. [CrossRef] [PubMed]
Vlodavsky, I.; Friedmann, Y.; Elkin, M.; Aingorn, H.; Atzmon, R.; Ishai-Michaeli, R.; Bitan, M.; Pappo, O.; Peretz, T.; Michal, I.;et al. Mammalian heparanase: Gene cloning, expression and function in tumor progression and metastasis. Nat. Med. 1999,
5, 793–802. [CrossRef]
Gingis-Velitski, S.; Zetser, A.; Kaplan, V.; Ben-Zaken, O.; Cohen, E.; Levy-Adam, F.; Bashenko, Y.; Flugelman, M.Y.; Vlodavsky, I.;
Ilan, N. Heparanase uptake is mediated by cell membrane heparan sulfate proteoglycans. J. Biol. Chem. 2004, 279, 44084–44092.
[CrossRef]
Levy-Adam, F.; Feld, S.; Cohen-Kaplan, V.; Shteingauz, A.; Gross, M.; Arvatz, G.; Naroditsky, I.; Ilan, N.; Doweck, I.; Vlodavsky, I. Heparanase 2 interacts with heparan sulfate with high affinity and inhibits heparanase activity. J. Biol. Chem. 2010,
285, 28010–28019. [CrossRef]
Katz, A.; Barash, U.; Boyango, I.; Feld, S.; Zohar, Y.; Hammond, E.; Ilan, N.; Kremer, R.; Vlodavsky, I. Patient derived xenografts(PDX) predict an effective heparanase-based therapy for lung cancer. Oncotarget 2018, 9, 19294–19306. [CrossRef]
Ferguson, V.L.; Simske, S.J.; Ayers, R.A.; Bateman, T.A.; Wang, H.T.; Bendele, A.; Rich, B.; Collins, D.; Scherrer, J.; Sennello, R.; et al.
Effect of MPC-11 myeloma and MPC-11 + IL-1 receptor antagonist treatment on mouse bone properties. Bone 2002, 30, 109–116.
[CrossRef]

Cells 2024, 13, 211

34.
35.

36.
37.

38.
39.
40.

41.
42.
43.
44.

45.
46.

47.
48.
49.
50.
51.

18 of 18

Alekseeva, A.; Mazzini, G.; Giannini, G.; Naggi, A. Structural features of heparanase-inhibiting non-anticoagulant heparinderivative Roneparstat. Carbohydr. Polym. 2017, 156, 470–480. [CrossRef] [PubMed]
Rohloff, J.; Zinke, J.; Schoppmeyer, K.; Tannapfel, A.; Witzigmann, H.; Mossner, J.; Wittekind, C.; Caca, K. Heparanase expression
is a prognostic indicator for postoperative survival in pancreatic adenocarcinoma. Br. J. Cancer 2002, 86, 1270–1275. [CrossRef][PubMed]Cohen, I.; Maly, B.; Simon, I.; Meirovitz, A.; Pikarsky, E.; Zcharia, E.; Peretz, T.; Vlodavsky, I.; Elkin, M. Tamoxifen induces
heparanase expression in estrogen receptor-positive breast cancer. Clin. Cancer Res. 2007, 13, 4069–4077. [CrossRef] [PubMed]
Hermano, E.; Goldberg, R.; Rubinstein, A.M.; Sonnenblick, A.; Maly, B.; Nahmias, D.; Li, J.P.; Bakker, M.A.H.; van der Vlag, J.;
Vlodavsky, I.; et al. Heparanase Accelerates Obesity-Associated Breast Cancer Progression. Cancer Res. 2019, 79, 5342–5354.
[CrossRef]
Kelly, T.; Suva, L.J.; Huang, Y.; Macleod, V.; Miao, H.Q.; Walker, R.C.; Sanderson, R.D. Expression of heparanase by primarybreast tumors promotes bone resorption in the absence of detectable bone metastases. Cancer Res. 2005, 65, 5778–5784. [CrossRef]
Maxhimer, J.B.; Pesce, C.E.; Stewart, R.A.; Gattuso, P.; Prinz, R.A.; Xu, X. Ductal carcinoma in situ of the breast and heparanase-1
expression: A molecular explanation for more aggressive subtypes. J. Am. Coll. Surg. 2005, 200, 328–335. [CrossRef]
Zahavi, T.; Salmon-Divon, M.; Salgado, R.; Elkin, M.; Hermano, E.; Rubinstein, A.M.; Francis, P.A.; Di Leo, A.; Viale, G.; deAzambuja, E.; et al. Heparanase: A potential marker of worse prognosis in estrogen receptor-positive breast cancer. NPJ Breast
Cancer 2021, 7, 67. [CrossRef]
Hammond, E.; Dredge, K. Heparanase Inhibition by Pixatimod (PG545): Basic Aspects and Future Perspectives. Adv. Exp. Med.
Biol. 2020, 1221, 539–565. [CrossRef] [PubMed]
Davies, B.; Morris, T. Physiological parameters in laboratory animals and humans. Pharm. Res. 1993, 10, 1093–1095. [CrossRef][PubMed]Zcharia, E.; Jia, J.; Zhang, X.; Baraz, L.; Lindahl, U.; Peretz, T.; Vlodavsky, I.; Li, J.P. Newly generated heparanase knock-out miceunravel co-regulation of heparanase and matrix metalloproteinases. PLoS ONE 2009, 4, e5181. [CrossRef]
de Boer, C.; Armstrong, Z.; Lit, V.A.J.; Barash, U.; Ruijgrok, G.; Boyango, I.; Weitzenberg, M.M.; Schroder, S.P.; Sarris, A.J.C.;
Meeuwenoord, N.J.; et al. Mechanism-based heparanase inhibitors reduce cancer metastasis in vivo. Proc. Natl. Acad. Sci. USA
2022, 119, e2203167119. [CrossRef] [PubMed]
Wood, L.D.; Canto, M.I.; Jaffee, E.M.; Simeone, D.M. Pancreatic Cancer: Pathogenesis, Screening, Diagnosis, and Treatment.
Gastroenterology 2022, 163, 386–402. [CrossRef]
Sung, H.; Ferlay, J.; Siegel, R.L.; Laversanne, M.; Soerjomataram, I.; Jemal, A.; Bray, F. Global Cancer Statistics 2020: GLOBOCAN
Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J. Clin. 2021, 71, 209–249. [CrossRef][PubMed]Daly, A.A.; Rolph, R.; Cutress, R.I.; Copson, E.R. A Review of Modifiable Risk Factors in Young Women for the Prevention ofBreast Cancer. Breast Cancer 2021, 13, 241–257. [CrossRef] [PubMed]
Yin, L.; Duan, J.J.; Bian, X.W.; Yu, S.C. Triple-negative breast cancer molecular subtyping and treatment progress. Breast Cancer
Res. 2020, 22, 61. [CrossRef]
Yap, T.A.; Aerts, J.G.; Popat, S.; Fennell, D.A. Novel insights into mesothelioma biology and implications for therapy. Nat. Rev.
Cancer 2017, 17, 475–488. [CrossRef]
Carbone, M.; Ly, B.H.; Dodson, R.F.; Pagano, I.; Morris, P.T.; Dogan, U.A.; Gazdar, A.F.; Pass, H.I.; Yang, H. Malignant
mesothelioma: Facts, myths, and hypotheses. J. Cell Physiol. 2012, 227, 44–58. [CrossRef]
Sekido, Y. Molecular pathogenesis of malignant mesothelioma. Carcinogenesis 2013, 34, 1413–1419. [CrossRef] [PubMed]

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury topeople or property resulting from any ideas, methods, instructions or products referred to in the content.