Electron cryotomography reveals ultrastructure

alterations in platelets from patients with

ovarian cancer
Rui Wanga,b, Rebecca L. Stonec, Jason T. Kaelberb,d, Ryan H. Rochata,b, Alpa M. Nickc, K. Vinod Vijayane,
Vahid Afshar-Kharghanc, Michael F. Schmida,b, Jing-Fei Dongf,g,h, Anil K. Soodc,i,j,1, and Wah Chiua,b,1
a
Graduate Program in Structural and Computational Biology and Molecular Biophysics, Verna and Marrs McLean Department of Biochemistry and
Molecular Biology, Baylor College of Medicine, Houston, TX 77030; bNational Center for Macromolecular Imaging, Verna and Marrs McLean Department of
Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030; cDepartment of Gynecologic Oncology, The University of Texas MD
Anderson Cancer Center, Houston, TX 77030; dDepartment of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030;
e
Department of Medicine, Baylor College of Medicine, Michael E. DeBakey Veterans Affair Medical Center, Houston, TX 77030; fCenter for Translational
Research on Inflammation, Michael E. DeBakey Veterans Affair Medical Center, Houston, TX 77030; gPuget Sound Blood Center, Department of Medicine,
School of Medicine, University of Washington, Seattle, WA 98104; hDivision of Hematology, Department of Medicine, School of Medicine, University of
Washington, Seattle, WA 98104; iDepartment of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030; and jCenter for
RNA Interference and Non-Coding RNA, The University of Texas MD Anderson Cancer Center, Houston, TX 77030

Contributed by Wah Chiu, September 30, 2015 (sent for review March 1, 2015; reviewed by Peter Frederik, Jun Liu, Kunle Odunsi, and David W. Scott)

Thrombocytosis and platelet hyperreactivity are known to be asso-
ciated with malignancy; however, there have been no ultrastructure
studies of platelets from patients with ovarian cancer. Here, we used
electron cryotomography (cryo-ET) to examine frozen-hydrated
platelets from patients with invasive ovarian cancer (n = 12) and
control subjects either with benign adnexal mass (n = 5) or free from
disease (n = 6). Qualitative inspections of the tomograms indicate
significant morphological differences between the cancer and con-
trol platelets, including disruption of the microtubule marginal band.
Quantitative analysis of subcellular features in 120 platelet electron
tomograms from these two groups showed statistically significant
differences in mitochondria, as well as microtubules. These structural
variations in the platelets from the patients with cancer may be
correlated with the altered platelet functions associated with ma-
lignancy. Cryo-ET of platelets shows potential as a noninvasive
biomarker technology for ovarian cancer and other platelet-related
diseases.
electron | cryotomography | platelet | microtubule | cancer
platelets in cancer pathology (6, 8) suggested the possibility of
ultrastructural perturbations in the platelets of patients with
ovarian cancer. These perturbations could provide structural
clues to improve our understanding of cancer-related hyperre-
activity, thrombocytosis, or as-yet-unknown platelet defects, and
could serve as potential biomarkers for screening for ovarian
cancer. Prior attempts at examining platelet ultrastructure in
diseases have been largely limited by methods that used plastic
embedding and chemical fixation (13–15). The present investi-
gation overcomes these technical limitations by using electron
cryotomography (cryo-ET) to visualize human platelets in their
naturally occurring pathophysiological states without chemical
fixation or staining (16).
Results
After approval by the institutional review boards for research
on human subjects, we prospectively collected peripheral blood
samples from patients with newly diagnosed invasive ovarian
cancer or benign adnexal masses (Table S1) before any chemo-
therapy or surgical treatment. Age-matched healthy females were

P latelets are small anucleate multifunctional cells derived

from megakaryocytes. Platelets circulate in the bloodstream
and respond to vascular lesions (1). In their resting state, they
adopt a discoidal shape and have an average lifespan of 5–7 d in
humans (2). Platelets have increasingly been recognized as playing
an important role in tumor growth and metastasis, in addition to
their traditional roles in hemostasis (3–6). Thrombocytosis
(platelet count >450,000/μL) is found in 31% of patients with
ovarian cancer and is associated with a poor clinical prognosis (7,
8). In addition, platelets in patients with cancer are functionally
altered, and often adopt a hyperreactive state (9). This platelet
hyperreactivity helps explain the higher thrombosis risk in these
patients (10). Furthermore, compelling preclinical and clinical
data demonstrate that platelets actively promote tumor growth
and metastasis through multiple pathways. Platelets form a phys-
ical shield to protect tumor cells from natural killer cell-mediated
lysis; they facilitate the adhesion of tumor cells to the endothe-
lium, allowing the critical extravasation step in the metastatic
cascade to occur; and they release a plethora of bioactive mole-
cules, including growth factors and cytokines stored in their se-
cretory granules, that promote angiogenesis and tumor cell growth
(11). It is shown in various experimental models that disruption of
platelet–tumor interactions abolishes these effects (5, 9), improv-
ing clinical outcomes of patients.
The relatively high incidence of thrombocytosis in these pa-
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Significance
Platelets are known to be both numerically and functionally
altered in some patients with cancer. However, structural dif-
ferences in the platelets from these patients have not been
studied. Here we use electron cryotomography to reveal that,
compared with control donors, the microtubule system and the
mitochondria of platelets from patients diagnosed with ovar-
ian cancer are significantly different. This finding suggests the
potential of electron cryotomography as a technology to de-
tect structural biomarkers of diseases affecting platelets.
Author contributions: M.F.S., J.-F.D., A.K.S., and W.C. designed research; R.W., R.L.S., V.A.-K.,
and M.F.S. performed research; R.W., R.L.S., A.M.N., K.V.V., and M.F.S. contributed new
reagents/analytic tools; R.W., J.T.K., R.H.R., M.F.S., J.-F.D., A.K.S., and W.C. analyzed data;
and R.W., J.T.K., V.A.-K., M.F.S., J.-F.D., A.K.S., and W.C. wrote the paper.
Reviewers: P.F., Maastricht University; J.L., University of Texas Medical School at Houston;
K.O., Roswell Park Cancer Institute; and D.W.S., Rice University.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
Data deposition: Representative tomograms of platelets in three different pathophysio-
logical states reported in this paper have been deposited in the EMDataBank, www.
emdatabank.org/ (accession nos. EMD-6471, EMD-6472, and EMD-6473).
1
To whom correspondence may be addressed. Email: wah@bcm.edu or asood@

mdanderson.org.
tient populations (12), which is related to cytokine signaling from This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
both tumor and nontumor tissue (8), and the implication of 1073/pnas.1518628112/-/DCSupplemental.

14266–14271 | PNAS | November 17, 2015 | vol. 112 | no. 46 www.pnas.org/cgi/doi/10.1073/pnas.1518628112

 recruited as controls. None of the participants had a primary
platelet disorder or a coexisting inflammatory condition, and
none was taking medications known to interfere with platelet
function. Average platelet counts and mean platelet volume of
the healthy control subjects were 279.6 × 103/μL and 7.9 fL, re-
spectively. Blood samples (anticoagulant: 0.38% sodium citrate
final concentration) were collected from the patients and healthy
subjects after informed consent was provided. Platelet-rich
plasma (PRP) was obtained by centrifugation and vitrified for
cryo-ET. From these frozen-hydrated samples, a total of 338 tilt
series of tomography images of individual platelets were gener-
ated. Each image series was then reconstructed into a 3D volume
called a tomogram. One hundred twenty tomograms with ade-
quate contrast were selected for filtering, feature annotation,
structural measurements, and statistical analysis. For the quanti-
tative analysis, we required at least five annotatable tomograms
from each subject. In addition, platelets from these subjects were
analyzed for aggregation induced by ADP.
Cryo-ET of Platelets from Healthy Control Subjects. We first exam-
ined platelets from the healthy controls (n = 6). Fig. 1A shows an
example slice from a 3D tomogram of a typical platelet of a
healthy subject (Movie S1). Fig. 2A shows the annotated structural
features of three platelets randomly selected from this healthy
group, including the plasma membrane, circumferentially coiled
microtubule (marginal band), α and dense granules, mitochondria,
and low-contrast vacuole-like (LCV) features. A fraction of the
LCV was seen as empty vacuole-like structures (Figs. 1A and 2A
and Movie S1). The rest of LCV exists as narrow and tortuous
invaginations of the surface membrane, the area of which was
impractical to estimate. We interpret this structural feature to be
the platelet surface-connected open canalicular system. Most
platelets from healthy subjects share a similar structural pattern.
The lack of a significant number of pseudopods indicates that the
observed platelets are mostly in the resting state. These structural
features are consistent with the morphology of platelets from
healthy subjects obtained by conventional 2D thin-section, plastic-
embedded electron microscopy (2, 13).
Cryo-ET of Platelets from Patients. Next, we examined platelets
obtained from women with either benign adnexal mass (n = 5) or
invasive ovarian cancer (n = 12). Qualitatively, platelets from
patients with benign masses (Figs. 1B and 2B) had a similar
appearance to those from the healthy subjects, including a rel-
atively intact microtubule ring and abundant secretory granules.
Fig. 1. Platelets from control subjects and patients with cancer, raw cryo-
electron tomogram. (A) Slice of a tomogram of a human blood platelet from
a healthy female donor. This platelet has a circumferential microtubule ring
and numerous secretory granules inside. The general shape is discoidal.
(B) Slice of a tomogram of a human blood platelet from a patient with a
benign tumor in the ovary. This platelet shares similar cytoplasmic structures
with the platelet in A, with the exception of the presence of an extended
pseudopod. (C) Slice of a tomogram of a human blood platelet from a pa-
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tient with invasive ovarian cancer. This platelet has more enlarged LCV
structures and an extended pseudopod. In contrast to the platelets in A and
B, this platelet has fragmented and fewer microtubules.
Wang et al.
Fig. 2. Platelets from control subjects and patients with cancer, annotated
tomogram. (A) Three randomly selected annotated platelets from healthy
donors. All of them have an intact marginal band of microtubules (blue)
enclosing most of their granules (α pink, dense green) and mitochondria
(red) inside. The plasma membrane is gray and the LCV is yellow. (B) Three
randomly selected annotated platelets from patients with benign masses.
Their structures are similar to those shown in A. (C) Three randomly selected
annotated platelets from patients with invasive ovarian cancer. Their mor-
phologies appear to be more heterogeneous compared with the six platelets
in the other two panels. They seem to have more LCV, fewer and shorter
microtubule filaments, and more mitochondria.
In contrast, platelets from patients with an invasive ovarian
cancer (Figs. 1C and 2C and Movie S2) had a strikingly different
appearance compared with those from either healthy subjects
(Figs. 1A and 2A) or patients with benign masses (Figs. 1B and
COMPUTATIONAL BIOLOGY
2B). In many of these platelets from patients with invasive cancer,
BIOPHYSICS AND
the microtubule marginal bands seen in the benign and control
platelets were severed and depolymerized to various degrees (Figs.
1C and 2C). Also unique to patients with invasive cancer was
marked visual and quantitative heterogeneity among individual
platelets, even those from the same patient (Fig. S1).
Quantification of Platelet Tomograms. To quantitate the structural
features of each platelet, we measured nine parameters (Figs. 3
and 4), including number of α granules, number of dense
granules, number of mitochondria, percentage of total platelet
area occupied by each of these three structures, microtubule
length, pseudopod number, and total platelet area. Tomograms
with insufficient contrast were excluded from further analysis.
Platelets from individual patients with ovarian cancer are het-
erogeneous (Fig. S1), which warrants the decision to measure
these parameters from at least five platelets per subject. These
nine parameters were chosen because they are associated with
platelet structural integrity and functions and are easily rec-
ognized in tomograms. For instance, α and dense granules in
our tomograms have relatively large size and characteristic
scattering densities because of their different biochemical
contents. The clinical utility of this study lies in the distinction
between cancer and noncancer groups. Therefore, we com-
bined the data from healthy donors and patients with benign
masses to form the control group for subsequent analyses be-
cause we found that many of these measurements exhibited the
same pattern for both healthy and benign samples, as expected
from qualitative analysis (Figs. 1 and 2). Measurements from
PNAS | November 17, 2015 | vol. 112 | no. 46 | 14267
 Fig. 3. Statistical analysis shows microtubule and mitochondria are discriminating parameters. (A) Microtubule strands are clearly seen in the subtomogram
of the platelet. Microtubule lengths are measured and compared between the control group (healthy donors plus patients with benign mass) and the cancer
group (patients with ovarian cancer). The cancer group has significantly fewer and shorter microtubules in their platelets than the control group. (B) Mi-
tochondria appear as enclosed double-membrane organelles; the inner membrane forms the characteristic cristae structure inside. Mitochondria numbers are
counted, and the percentage of the whole platelet area covered by mitochondria is measured. The cancer group has significantly more mitochondria in their
platelets compared with the control group. They also have a higher percentage of area covered by mitochondria.

this control group (n = 11) were compared with those from
patients with invasive ovarian cancer (n = 12). Among these
measurements, three of the nine parameters were found to
show significant differences between control and cancer subjects:
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microtubule length, mitochondria number, and percentage of
platelet total area occupied by mitochondria (Fig. 3). Again, these
three parameters did not differ significantly between the benign
and healthy donors.

Fig. 4. The six other measured platelet parameters are found not to be significantly different between the control and cancer groups. These are (A) platelet area,
(B) number of pseudopods, (C) number of α granules and percentage of granule area, and (D) number of dense granules and percentage of granule area.

14268 | www.pnas.org/cgi/doi/10.1073/pnas.1518628112 Wang et al.

 Our quantification shows that the mean microtubule length was
significantly shorter in platelets from patients with invasive ovarian
cancer compared with those from the control group (36.08 ±
9.70 μm vs. 66.17 ± 14.42 μm; P = 0.0001; Fig. 3A). Qualitatively,
microtubules of the patients were fragmented, instead of forming
intact marginal band rings as in the control group (Fig. 2). Because
all the quantifications were performed by the same investigator, it
is likely that the analysis was self-consistent, but not necessarily
unbiased. However, subjectivity is reduced because we chose to-
mograms having unequivocally good contrast for analysis.
We also found differences in the number of mitochondria and
the percentage of the whole platelet area covered by the mito-
chondria. Platelets from patients with cancer had more mito-
chondria than platelets from the control group (Fig. 3B) (7.26 ±
2.44 vs. 4.85 ± 1.46 mitochondria per platelet; P = 0.01). The
absolute number of mitochondria per platelet in the control group
is consistent with previous reports (17). The mitochondria from
patients with cancer occupied a larger fraction of the area of the
platelet (3.91 ± 1.21 vs. 2.47 ± 1.18% of platelet area; P = 0.009).
Most of the differences in total mitochondria area among platelets
can be explained by differences in the number of mitochondria per
cell (R2 = 0.80; n = 120).
The other six quantitated parameters did not show statistically
significant differences (Fig. 4).
Predictive Modeling. To assess whether cryo-ET quantification
provides enough information to predict whether a subject has a
malignancy, and to assess whether the sample size in this study
was sufficient to provide predictive power, we performed dis-
criminant analysis with leave-one-out cross validation (18). Pre-
diction of malignancy was made using only the number of
mitochondria and the microtubule length. In 20 of 23 cases, the
malignancy status was correctly predicted, yielding an accuracy
of 87% in this study.
Platelet Aggregation Assay. Platelet–platelet and platelet–leuko-
cyte aggregates are often detected in peripheral blood of patients
with inflammatory conditions. These aggregates are formed among
primed and activated platelets, and their presence is widely rec-
ognized as a risk for thrombosis. In addition to the detectable
structural differences between control and cancer platelets, we
also investigated their functional consequences by measuring
platelet aggregation induced by ADP at 2.5, 5, and 10 μM. Using
lower concentrations of ADP allows us to detect platelet hyper-
reactivity, which was defined as ≥30% aggregation induced by
2.5 μM ADP. In a previous study of 359 healthy subjects, ADP at
1.5–2.5 μM was used to differentiate hypo- from hyperreactive
platelets (19). This concentration of ADP does not induce sig-
nificant platelet aggregation in most healthy subjects. Our data
show that mean levels of platelet aggregation induced by 2.5 and
5 μM were significantly higher in the patients with ovarian cancer
compared with controls (Fig. 5A). We also found that 75% of
patients with ovarian cancer and 27% of our controls have hy-
perreactive platelets, as defined by the above criterion (Fig. 5B),
indicating an enhanced response of cancer platelets to the lowest
dose of ADP. At the highest concentration of ADP (10 μM), the
two groups become indistinguishable in their aggregation, sug-
gesting the platelets from patients with ovarian cancer are not
activated before the addition of the agonist.
Discussion
Thin-section electron microscopy has revealed the morphologic
features of platelets associated with a variety of (patho)physio-
logical conditions (15), but this imaging method lacks 3D in-
formation of the subcellular contents in a platelet close to its
native state (16). Cryo-ET has emerged as a viable technology to
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study well-preserved ultrastructure of mammalian and bacterial
cells in 3D (20–22). Therefore, we used cryo-ET to examine the
Wang et al.
Fig. 5. Platelet aggregation assay shows a correlation between hyperac-
tivity and malignancy. (A) The mean level of platelet aggregation is signif-
icantly higher in patients with invasive ovarian cancer compared with
control subjects (Student’s t test, n = 23). In comparison, there is no signif-
icant difference in maximal aggregation between the two groups. (B) There
are significantly more patients with invasive ovarian cancer with hyperre-
active platelets (black bar vs. normal platelets indicated by white bar), as
defined by aggregation at 2.5 μM ADP (χ2 test, P = 0.018), but not at 5 and
10 μM of ADP compared with control subjects. CTR, control group; MT,
metastatic tumor.
structures of individual platelets from patients with ovarian tu-
mors. The acquired tomograms of platelets are rich in 3D
structural information, allowing the subcellular arrangement of
various components in different physiological states to be seen
for the first time to our knowledge.
The platelets from the healthy subjects studied here are mostly
in the resting state, as evidenced by the paucity (0.4 ± 0.7 per
platelet) of extended pseudopods called filopodia, the presence of
which is a structural landmark of an activated platelet. Because
the platelet is crowded with numerous subcellular components, we
only annotated the most recognizable features in this study.
Platelets from patients with cancer share many visually identifiable
subcellular features with platelets from control subjects (Fig. 4).
More than 70% of α granules in all platelets were nearly spherical;
the rest were irregularly shaped and morphologically heteroge-
COMPUTATIONAL BIOLOGY
neous (23). The mean diameter of the near-spherical α granules
BIOPHYSICS AND
was 221.34 ± 56.03 nm, in agreement with previously reported
sizes of 200–500 nm by other imaging technologies (24). The
numbers of α and dense granules per tomogram for the control
platelets were 43.0 ± 22.9 and 5.5 ± 1.9, respectively, which is
consistent with reported counts of 50–80 and 3–9 per platelet (25).
Platelets from patients with cancer appeared qualitatively to have
a higher percentage area covered by enlarged LCV structures,
which are likely to be either enlarged open canalicular system or
empty granules, compared with those from healthy subjects and
patients with benign masses (Figs. 1C and 2C).
To quantify the visual differences of the platelets, we mea-
sured the length, area, and/or number of the observed sub-
cellular features. Each tomogram has a missing wedge resulting
from limited specimen tilt angles, resulting in inaccuracy in
the measurements along the direction of the electron beam.
Therefore, our size quantifications of the identified features
are made from their central areas instead of the volumes. Our
analysis shows the mean platelet area did not differ significantly
between control subjects and patients with cancer. However,
three other quantitative measures showed significant difference
between control and cancer platelets including microtubule
length, the number of mitochondria, and the percentage of the
total platelet area occupied by mitochondria.
Platelets contain fully functional mitochondria that are active in
ATP production, regulation of redox signals, and platelet apo-
ptosis (26). With cryo-ET, mitochondria are easily distinguishable
in the crowded cytosol from other membranous organelles be-
cause of their double membrane and internal cristae. A significant
PNAS | November 17, 2015 | vol. 112 | no. 46 | 14269
 increase in the number of, and percentage of platelet area covered
by, mitochondria (Fig. 3B) suggest they may serve as one of the
structural signatures to differentiate platelets of patients with in-
vasive cancer from those of the control subjects. The number of
mitochondria per platelet is elevated by about 50% among pa-
tients with malignancies, with only a small increase (<15%) in the
mean size of a mitochondrion.
The biological cause for the increase in mitochondria remains
further to be investigated. Activated platelets can release mito-
chondria in the form of microparticles (17), and mitochondria may
replicate in circulating platelets (27). We occasionally observed
small “platelets,” which may represent platelet microparticles,
whose size can approach that of a true platelet (Fig. S1). Never-
theless, it is likely that platelet mitochondrial number is largely
determined in the megakaryocyte during thrombopoiesis. Pro-
thrombopoietic factors affecting the megakaryocyte are emitted by
tumor and host tissue during ovarian malignancy. This dysregulation
can cause thrombocytosis. However, thrombocytosis is observed in
only a third of patients at initial diagnosis, which limits its diagnostic
potential (8). Platelet mitochondrial number warrants further in-
vestigation as a potentially more sensitive marker of megakaryocytic
dysregulation, and because platelet activity affects disease progres-
sion in an animal model (8).
In addition, we also detect a significant reduction in the length
of microtubules. Microtubules are critical for maintaining the
platelet’s discoidal shape and participate in platelet shape changes
and granule movements upon platelet activation (2).
Platelet hyperreactivity, which has been reported in cancer and
other diseases of inflammation but has not been morphologically
explained, could be caused by the disintegration of microtubules
(Fig. 3A), and in particular the disruption of the marginal band
(Fig. 2 and Fig. S1). Platelet hyperreactivity means platelets are
primed to activation: they form aggregates when exposed to lower
concentrations of agonists than normal platelets, which require
higher concentrations of agonists to activate. Hyperreactive plate-
lets are thus not activated platelets, but they become activated
more easily upon stimulation. This hyperreactivity of platelets from
patients with cancer is supported by the aggregation assay (Fig.
5A). Nine of our patients with cancer (75.0%) were identified as
having hyperreactive platelets (Fig. 5B) compared with three of our
control subjects (27.3%; P = 0.039). There may be an underlying
mechanism of cytoskeleton dysregulation in ovarian cancer causing
both hyperreactivity and reduction in microtubule length that re-
quires further study.
Abnormal platelet number (thrombocytosis) and hyperreactivity
of platelets have been reported in cancer (8, 28), but have not
been useful as screening tools because of their low positive pre-
dictive values. Our cryo-ET analysis demonstrates that the cancer
group has more fragmented microtubules and more mitochondria
in their platelets compared with the control group. In this study,
the presence or absence of a malignancy was predicted with 87%
accuracy solely from information contained within the cryotomo-
grams of subjects’ platelets. These findings suggest a structural
basis for the altered platelet function associated with malignancy,
and indicate that platelet ultrastructure could be useful as a bio-
marker for ovarian cancer detection.
To further substantiate our proof-of-principle observations
before becoming a clinical tool, larger patient numbers and a
higher-throughput protocol will be required. Recent advances in
cryo-ET suggest it can be used as a high-throughput method in a
research laboratory (29–31), raising the possibility of its use in
clinical laboratory. The increase in cell tomogram contrast using
the Zernike phase plate technology in cryo-ET will likely ease
the recognition and quantification of the observed subcellular
features (32). Software algorithms are being actively developed
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for automatic feature extraction of filamentous features (33).
Therefore, the ability of cryo-ET to measure fine ultrastructure
14270 | www.pnas.org/cgi/doi/10.1073/pnas.1518628112
aberrations opens the possibility for this technology to be used for
large-scale biomarker development in this and other disease states.
Methods
Cryo-ET of Human Platelets. PRP was separated from drawn blood samples and
then vitrified for cryo-ET. Women with benign adnexal masses had a final
pathological diagnosis of ovarian cystadenoma/fibroma (n = 4) or corpus
luteum cyst (n = 1). Protocol was approved by Baylor College of Medicine
Institutional Review Board, protocol no. H-12278. Patients with ovarian
cancer had stage III–IV high-grade serous ovarian cancer or other ovarian
malignancies (n = 12). All patients provided written informed consent for
participation in this study. All samples were obtained preoperatively, before
any chemotherapy treatment. Blood (2.5 mL) was drawn using a 21-gauge
needle Vacutainer brand blood collection set (Becton Dickinson) into 3.8%
(wt/vol) sodium citrate polyethylene tubes. The blood was immediately
centrifuged at 150 × g for 20 min, and the PRP was collected. Blood and PRP
were maintained at room temperature during manipulation. Quantifoil
holey carbon supported transmission electron microscope grids with 3.5-μm
circular holes (Quantifoil Micro Tools GmbH) pretreated with colloidal gold
(fiducial tracer, 15 nm) were glow-discharged (15 s) before use. PRP (3 μL)
was applied to the grid and then blotted with calcium-free blotting paper,
using a Vitrobot (Mark IV, FEI Corp), and immediately plunged into liquid
ethane at liquid nitrogen temperature to vitrify the platelets (1 blot, 3-s
wait, or 2 blot, 2-s wait).
All platelet samples were vitrified for cryo-ET study within 1 h of blood draw
to prevent any potential activation that may disturb the platelet ultrastructure.
The frozen grids with platelets were then transferred at liquid nitrogen
temperature into JEOL electron cryomicroscopes (JEM 2100, JEM 2200FS, or
JEM 3200FSC; JEOL Ltd.) for imaging. Low-dose conditions were used to
preserve the structural integrity. For JEM2200FS and JEM3200FSC micro-
scopes, an in-column energy filter (slit = 15 eV) was applied to enhance the
image contrast by zero loss imaging. Using the SerialEM package (34), tilt
series were recorded at 200 keV/300 keV on a Gatan 4,096 × 4,096 pixel CCD
camera (Gatan Inc.). Images were collected at a defocus range of ∼8–15 μm
and microscope magnification range of 8–12,000×. Total electron dose per
tomogram was ∼75 electrons/Å2, as typically used for cryo-ET (35). Each tilt
series has 62–66 CCD frames with a fixed 2-degree increment.
A total of 338 tilt series of individual platelets were recorded in this study.
For each individual under study, 10–20 platelets at suitable places for im-
aging (e.g., not close to the grid bar) were selected without a particular bias,
imaged, and processed. Platelets were selected for imaging on the basis of
their positions on the microscopy grid, and not on internal features that are
not visible before 3D reconstruction. Of these 10–20 platelet tomograms, at
least five tomograms had adequate contrast to clearly visualize microtubules
and to distinguish double-membrane mitochondria from single-membrane
α granule. In general, quantifiable tomograms were randomly selected for
quantification. Some subjects yielded fewer than five visually good tomo-
grams, and were not included for further tomographic reconstruction and
statistical analysis. The final data set of 120 tomograms was selected from
healthy female controls (n = 6), women with benign adnexal masses (n = 5),
and patients with newly diagnosed invasive ovarian cancer (n = 12).
Platelet Tomogram Processing and Quantification. Image stacks were aligned
with gold tracers, using the IMOD (version 4.0.27) package (36), yielding 3D
reconstructed tomograms (voxel 4k × 4k × 1k, ∼11–13 Å per pixel sampling).
To enhance the contrast, tomograms were averaged by two and filtered by
denoising tools such as low-pass and nonlinear anisotropic diffusion (37) in
the EMAN2 (38) and IMOD packages. Structural features such as α granules,
dense granules, mitochondria, microtubules, and membrane systems were
identified and manually annotated using Amira (version 5.2; Visage Imaging
Inc.). Microtubules were clearly seen as a coiled circumferential marginal
band in the control group and as partially fragmented filaments in some
platelets in the cancer group. Granules with very dark densities were an-
notated as dense granules. Single membrane-bound granules with gray
densities were annotated as α granules. Organelles with double membranes
were annotated as mitochondria. The inner membrane of mitochondria was
seen to form cristae-like structures.
Structural features (microtubule length, α granule number, α granule
area, dense granule number, dense granule area, mitochondria number,
mitochondria area, pseudopod number) were manually quantified for each
individual platelet tomogram, using Amira. The number of pseudopods,
α and dense granules, and mitochondria was counted with IMOD. The 3D
tomogram of the whole platelet was projected in Z direction, and the area
of the whole platelet enclosed by the plasma membrane in the projection
image was measured as platelet area. A small fraction of platelets in the
Wang et al.
 cancer group had part of their platelet area beyond the CCD frame. For
these platelets, the area of the polygon was measured as an approximation
for the whole platelet area. For mitochondria, the area of their central slice
was measured individually in each tomogram and summed up as the area
of mitochondria. The mitochondria area was then divided by the whole
platelet area to estimate the percentage of mitochondria area. α and dense
granule areas were measured in the same way. A small fraction of α and
dense granules adopt an irregular shape compared with the spherically
shaped majority. Irregularly shaped granules were projected in the Z di-
rection first, and their areas in the projection image were measured. For
microtubules, a mask was drawn in their central slice and then was corrected
by an algorithm to remove redundant annotation (i.e., measurement of the
same microtubule on consecutive sections of the tomogram; Movie S3), and
the total area of the mask was measured in Amira. The algorithm to remove
redundant microtubule mask is a Python script using two functions that was
applied offline to the manually selected 3D mask that was produced by
AMIRA. This area was then divided by 250 Å (the microtubule diameter) to
estimate the microtubule length (Fig. 3). No change in significance was
observed for any comparison when the redundancy-removing algorithm was
omitted. All the quantification was performed by the same researcher to
maintain the consistency for the entire study.
Statistical Analysis of Quantified Platelet Tomograms. Unpaired, two-tailed
Student t test was performed to test for statistically significant differences in
the measured features in the platelet tomograms between the two groups
(control group that comprises healthy donors and patients with benign mass;
cancer group that comprises patients with ovarian cancer), with a P < 0.05
considered to be significant.
Predictive Modeling. We evaluated the potential of cryo-ET to distinguish between
subjects with or without malignancy. Each subject’s disease state was coded as
malignant (n = 12) or nonmalignant (n = 11). Number of mitochondria and total
microtubule length were selected as predictors for discriminant function analysis
based on the qualitative observations of the individual who performed
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ACKNOWLEDGMENTS. This research has been supported by NIH Grants
P41GM103832, HL071895, HL085769, HL081613, and CA177909; Department
of Defense Grants OC120547 and OC093416; an Ovarian Cancer Research
Fund Program Project Development Grant; the Bettyann Asche Murray
Distinguished Professorship; and a Baylor College of Medicine and MD
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PNAS | November 17, 2015 | vol. 112 | no. 46 | 14271