Vol. 98 No.

6 December 2004

ORAL AND MAXILLOFACIAL PATHOLOGY Editor: Alan R. Gould

Application of laser capture microdissection to phage display peptide library

screening
Hai Lu, MD,a Dadi Jin, MD,b and Yvonne L. Kapila, DDS, PhD,a,c San Francisco, Calif,
Guangzhou, China, and Ann Arbor, Mich
UNIVERSITY OF CALIFORNIA SAN FRANCISCO, NANFANG HOSPITAL, AND UNIVERSITY OF MICHIGAN

Objective. When identifying important regulatory genes using methods such as phage display peptide library screening it is
critical to select such peptides from cells and tissues in their native state. Here, we report a novel approach to screen tumors
using phage display and laser capture microdissection (LCM).
Study design. A phage peptide library was screened directly on fresh oral tumor tissue, such that specifically bound peptides
were selected from fresh tumor cells in the native tissue state. Tissue processing conditions were modified to ensure the survival
of the bacteriophage.
Results. Our results demonstrate that live phage-peptide conjugates can be recovered from laser capture microdissected cells in
a form suitable for additional cycles of amplification.
Conclusion. Thus, LCM will be a valuable adjunct to phage display studies.
(Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;98:692-7)

Peptides that recognize specific cell types promise to be
valuable tools in research and clinical applications. In
preclinical animal studies, anticancer drugs conjugated
to tumor-targeting agents generally display greater anti-
tumor efficacy than the respective free drugs. In clinical
studies, however, the use of monoclonal antibodies or
peptides in the targeting of antitumor agents has proven
less successful.1 One potential reason for this clinical
failure may be that current phage display peptide library
screening is usually performed against purified cell-
surface markers, whole cells in tissue culture, or human
tissues xenografted into animals, instead of directly on
human tissues.2 The cellular environment and extracel-
This work was supported by the UCSF Comprehensive Cancer Center/
Stewart Trust Research Award Program, ST-04-03 (to Y.L.K.).
a
Postdoctoral Fellow, Department of Stomatology, School of Dentistry,
University of California San Francisco, San Francisco, Calif.
b
Professor, Nanfang Hospital, Guangzhou, China.
c
Associate Professor, University of Michigan, School of Dentistry,
Department of Periodontics/Prevention/Geriatrics, Ann Arbor, Mich;
Assistant Professor, Department of Stomatology, School of Dentistry,
University of California San Francisco, San Francisco, Calif.
Received for publication Aug 9, 2004; returned for revision Aug 9,
2004; accepted for publication Sep 10, 2004.
1079-2104/$ - see front matter
Ó 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.tripleo.2004.09.004
lular matrix (ECM) play important roles in gene expres-
sion. Therefore, when methods such as phage display
peptide library screening are used to identify important
proteins, it is critical to select such peptides from cells in
their native state. In addition, selecting peptides that bind
specifically to cell-surface proteins responsible for tu-
mor invasion is important for targeted therapy. Compar-
ing cell-surface proteins in the center of the tumor with
those in the invasive rim may provide insights into
protein expression patterns that are important for this
process.3 However, it has been recognized that regard-
less of tumor type, bulk tissue, even in its highest purity,
still has potential contamination from stromal, endothe-
lial, and inflammatory cells.4 Thus, the problem of cellu-
lar heterogeneity has been a significant barrier to the
molecular analysis of normal and tumor tissue.
Here we report a novel approach for screening tumors
in their native environment. We used a combination of
laser capture microdissection (LCM) and random phage
peptide library screening (Fig 1). This novel method
enabled us to directly screen a phage peptide library on
fresh oral tumor tissue and recover specific cell-binding
peptides from tumor cells harvested by LCM. LCM is
a rapid and reliable method for procuring pure
populations of targeted cells or cell groups from
a section of complex, heterogeneous tissue.5,6 LCM is

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Volume 98, Number 6 Lu, Jin, and Kapila 693

Fig 1. Panning with a phage display peptide library. Phage display describes a selection technique in which a peptide is expressed
as a fusion with a coat protein of a bacteriophage, resulting in display of the fused peptide on the exterior surface of the phage
virion, while the DNA encoding the fusion resides within the virion. Phage display has been used to create a physical linkage
between a vast library of random peptide sequences to the DNA encoding each sequence, allowing rapid idenfication of peptide
ligands for a variety of target molecules by an in vitro selection process called ‘‘panning.’’ Panning is carried out by incubating
a library of phage-displayed peptides with the target, washing away the unbound phage, and eluting the specifically bound phage.
The eluted phage is then amplified and taken through additional cycles of panning and amplification to enrich the pool of phage in
favor of the tightest binding sequences. After 3 to 4 rounds, individual clones are characterized by DNA sequencing and ELISA.

based on the adherence of visually selected cells to MATERIALS AND METHODS

a transparent thermoplastic film (ethylene vinyl acetate Random phage peptide library
polymer), which prevents the tissue sections from being PhD-C7C phage peptide library CX7C (C = cysteine;
directly shot by the low-energy infrared laser pulse and X = any amino acid) and empty M13KE vector were
protects the phages from the burning laser beam. purchased from New England Biolabs, Inc (Beverly,

694 Lu, Jin, and Kapila
Mass). The titer of the library was ;1012 plaque-
forming units/mL (pfu/mL). Purification and amplifica-
tion of phage particles was performed according to the
manufacturer’s protocol.
Tissue processing and sectioning
Fresh human oral squamous cell carcinoma (SCC:
HSC-3 cell) tissue was resected from host nude mice.7
After resection, the tissues were cut into 6 3 6 3 6 mm3
blocks, immediately incubated in a 1-mL 2 3 1011 pfu/
mL phage-peptide library, specific phage-peptides, or
M13KE control solution (Dulbecco Modified Eagle’s
Minimal Essential Medium containing 200:1 diluted
Protease Inhibitor Cocktail [Sigma, St Louis, Mo] and
1% bovine serum albumin), rocked gently for 30 minutes
and 1, 2, 5, 7, and 10 hours at 4 8C. The tissues were then
washed in phosphate-buffered saline (PBS) 3 times,
embedded in Optimal Cutting Temperature (OCT)
compound (Sakura Finetek, Torrance, Calif), and frozen
in liquid nitrogen. Frozen sections (10 mm thick) of both
tumor and normal mucosal tissue were cut on a cryostat
(Leica, Milton Keynes, Buckinghamshire, UK), mounted
onto clean uncoated glass slides, and stored at ÿ808C
until further processing. Institutional Review Board
approval was obtained for these studies from the
University of California San Francisco Committee on
Human Research.
Tissue staining and dehydration by freeze drying
Frozen tissue sections were thawed at room temper-
ature for 30 seconds, immersed in 70% ethanol for 30
seconds, and stained with hematoxylin and eosin.3,8
Briefly, sections were dipped 3 times in deionized
water, incubated in Mayer’s hematoxylin (Sigma) for
1 minute at room temperature, dipped 5 times in TBST
(tris (hydroxymethyl) aminomethane-buffered saline with
0.1% Tween-20), incubated in an ammonium hydroxide
(Fisher Scientific, Santa Clara, Calif) solution (0.18% in
PBS, pH 7.0) for 30 seconds, dipped 5 times in TBST,
and incubated in 0.5% aqueous eosin (Sigma) for 30
seconds at room temperature. The sections were rinsed
another 5 times with ice-cold TBST to remove non-
specifically or weakly bound phages. After staining, the
sections were incubated in a cryoprotectant (PBS with
9% sucrose) for 2 minutes at room temperature. The
sections were dried on dry ice for 2 hours under high
pressure (200 mtorr) using a mobile freeze-dryer (Virtis
Company Inc, Gardiner, NY), then warmed to room
temperature. The tissue sections were also dried for 1, 2,
3, 5, or 10 days to determine the effect of drying time on
the frozen samples and on live phage recovery.
LASER CAPTURE MICRODISSECTION
After freeze drying, LCM was performed using
CapSur transfer film with the PixCell II LCM system
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December 2004
(Arcturus Engineering, Inc, Mountain View, Calif). Cells
of interest were identified and harvested from the SCC
tumor islands and invasive tumor rims. About 600 SCC
cells (400 LCM pulses using a 30-mm beam diameter and
1 ms per pulse) were captured from each of the tissue
samples. As a negative control and to measure the amount
of nonspecific phage contamination when transfer film is
overlayed on the tissue sections, another CapSure transfer
film was overlayed on sections for the same amount of
time, then retrieved but without LCM.
Phage recovery from microdissected tissues
After microdissection, the cells attached to the
CapSure transfer films were placed in 500 mL Eppendorf
microcentrifuge tubes (Applied Biosystems, Foster City,
Calif) with 100 mL of the host bacterial solution
(OD600 = 0.5), vortexed, and incubated at 37 8C for 20
minutes. The pfu number or amount of phage recovered
for each sample was determined by serially diluting the
sample and subsequently plating these dilutions on
Luria-Bertani/isopropylthio-beD-galactoside/5-bromo-
4-chloro-3-indolyl-beD-galactoside agar plates.
Immunohistochemistry
For immunohistochemical detection of binding with
phage-peptides or M13KE control phages, frozen
sections were fixed in 70% ethanol for 30 seconds,
then phage proteins were detected in the tissues by
immunostaining as described.7 Briefly, nonspecific
binding was blocked with a 2% nonfat milk solution in
PBS for 15 minutes at room temperature, then the
sections were incubated in a 1:500 dilution of a horse-
radish peroxidase (HRP)-conjugated mouse anti-M13
monoclonal antibody (Amersham Pharmacia Biotech
Inc, Piscataway, NJ) for 30 minutes. Staining controls
were evaluated with a nonimmune mouse immunoglob-
ulin-G (IgG) antibody. The substrate solution used was
1 mg/mL of diaminobenzidine (DAB) in PBS with 0.1%
hydrogen peroxide. Then, the sections were counter-
stained with 0.5% hematoxylin.
RESULTS AND DISCUSSION
Fresh human oral SCC tissue and normal mucosal
(epithelium, blood vessels, and adjacent connective tis-
sue) tissue sections were incubated and processed in the
phage peptide library solution. The sections were rinsed
to remove nonspecifically or weakly bound phages and
dehydrated by freeze-drying (20 mtorr) for up to 10
days. The phage-bound cells of interest were then
selected by LCM, and the specific binding phages were
recovered by transfection into host bacteria. The phages
eluted from LCM-harvested cells were amplified and
used as the input phage sublibrary for the next round of
biopanning. This procedure was repeated twice.
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Volume 98, Number 6 Lu, Jin, and Kapila 695

Fig 2. Selective binding of phage to tumor after 3 rounds of selection. After each round, the selected phages were amplified and
used to assess the specificity of phage binding to SCC tumor tissue. After nonspecifically or weakly bound phages were washed
away, the whole sections were scraped from the slides, and phages were recovered by transfection into host bacteria. The tumor/
mucosa ratio (number of phages recovered from tumor divided by number of phages recovered from the same amount of mucosa)
was about 12:1 after 3 rounds of biopanning. The number of phages (transducing units, TU, y axis) recovered from each section
varied to some extent, but the tumor/mucosa was consistent across experiments. The bars show standard error of the mean (SEM)
from plating in triplicate.

Fig 3. Graphs illustrate the effects of ethanol (A) and freeze-drying (B) on phage survival. A, The sections, incubated with phages,
were fixed in 70% ethanol. The toxic effect of ethanol on phages was time dependent; that is, the longer the exposure to ethanol the
more toxic to the phages. For performing LCM, 30 seconds of tissue fixation with ethanol was sufficient and only 29% of phages
were killed with this protocol. B, The sections, incubated with phages, were dried for 1, 2, 3, 5, and 10 days. The results
demonstrate that freeze-drying did not appreciably alter phage survival for up to 10 days. Approximately, 75% to 85% of phages
survived with this protocol. Each value represents the mean of 3 experiments performed in triplicate.

After the second and third rounds of selection, tumor
and mucosa tissue sections were washed to remove
nonspecifically bound phages, and the binding specific-
ity of phages selected from the previous round was
assessed. Analysis of the phage numbers recovered from
each whole section showed the phage solutions re-
covered from the second and third rounds had 8-fold and
12-fold greater affinity, respectively, for SCC tumor
tissue than for normal mucosa (Fig 2).
One of the most important steps for successful LCM
is the tissue dehydration step. However, we found that
prolonged and repeated exposure to ethanol and xylene
killed almost all the phages in our tissue samples.
Therefore, we tested and used a freeze-drying protocol.
Freeze drying has been used to preserve proteins and
microorganisms for extended periods.9 Freeze drying
did not appreciably alter phage survival for up to 10 days
(Fig 3, A). In addition, longer drying times facilitated the
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696 Lu, Jin, and Kapila December 2004

Fig 4. Use of LCM to selectively harvest an SCC tumor island (A), the SCC tumor rim (B), and a control lymph node (C). Image
magnification 10 3 10. The arrows point to tumor cells that will be targeted for LCM retrieval (Pre-LCM). Tissues attached to the
LCM transfer films were removed from the heterogeneous tissues (Post-LCM). The harvested cells were transferred to the transfer
film as illustrated (Cap). These 7-mm frozen sections were counterstained with H/E before LCM retrieval to illustrate the detailed
histomorphology of the SCC tissue. After H/E staining, the sections were washed extensively in cold TBST, incubated in a sucrose
cryoprotectant, then freeze dried for 48 hours under high pressure (20 mtorr). This processing technique likely altered the acid/base
characteristics of the stain and also likely washed some of the H/E stain away. LCM was then performed with CapSure transfer film
and the PixCell II LCM system (Arcturus Engineering, Mountain View, Calif). About 600 SCC cells (400 1-ms LCM pulses with
a 30-mm beam diameter) were captured from each of the tissue samples. Phages recovered from the tumor cells were amplified and
reincubated with fresh tissues in 2 consecutive rounds.

dissection and retrieval of cells and tissues by LCM.
Furthermore, freeze drying optimally maintains the 3-
dimensional structure of proteins, thus also allowing for
improved protein research via LCM in future studies.
Another step that may be toxic to bacteriophages is
fixation in 70% ethanol. However, we found that more
than 70% of the phages survived a 30-second incubation
in 70% ethanol at 48C (Fig 3, B). To avoid additional
exposure of the tissue to ethanol, we used an aqueous
eosin solution for staining rather than an ethanol
solution. These measures allowed successful LCM and
retrieval of live phages.
The major goal of the present study was to develop
a phage peptide library biopanning method suitable for
use directly on fresh tissue. To obtain fresh tissue easily
and to set up strict parallel controls, we used human
tumor tissue xenografted in nude mice rather than fresh
human tumor tissue. However, the findings in this study
demonstrate that our method is suitable for screening of
a phage peptide library directly on fresh human biopsy
samples and rescuing peptide sequences from tumor
cells in their native environment or from different cell
groups within the same organ (Fig 4).
Previous authors10,11 reported that after injection of
a phage peptide library solution intravenously in mice,
the phage peptide spread outside the blood vessels and
into the tumor tissues. In the present study, fresh tissue
blocks were incubated in the phage peptide library
solution with gentle rocking for 7 hours at 48C. After the
frozen sections were washed with TBST 10 times,
a strong phage protein signal was still identified.
Immunohistochemical analysis showed that the phage
protein could penetrate into tumor tissue from both the
edge of the tumor and the blood vessels inside the tumor
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Volume 98, Number 6 Lu, Jin, and Kapila 697

Fig 5. Immunohistochemical detection of phage that has penetrated fresh tumor tissue. Fresh SCC tissue (6 3 6 3 6 mm)
was incubated in a 2 3 1011 TU phage-peptide library solution at 48C for 7 hours. After incubation, frozen sections were prepared
and stained immunohistochemically with horseradish peroxidaseeconjugated mouse anti-M13 monoclonal antibody (1:500,
Amersham Pharmacia, Piscataway, NJ) to identify phage proteins and phage route of entry into tissues. No biopanning was done
and sections illustrate the route of entry into the tissues by the phage. Brown staining represents the phage protein signal; blue
indicates counterstaining of cell nuclei with hematoxylin. A, Penetration of phage from the edge of the tissue. B, Penetration of
phage into the tissue from a blood vessel (arrow). Image magnification 10 3 20.

tissue (Fig 5, A, B). Our findings show that phages
selected by the novel method described here penetrate
tumor tissues and bind to tumor cells. Thus, peptides
selected in this fashion may be clinically useful for
delivery of intravenously administered drugs to specif-
ically targeted tumor cells.
Although the random peptides selected may not
directly identify the target protein, the peptides can be
sequenced and this information can be used to scan
protein or gene sequence banks and/or used to design
probes to scan gene libraries for identification of the
target protein or receptor. Once identified in this manner,
the target protein or receptor can be further tested for
specificity on tumor tissues. In summary, this short
communication on proof of concept illustrates that ap-
plication of laser capture microdissection to phage lib-
rary screening is possible, however further studies are
needed to identify the specific peptides in question.
The authors thank Dr Randall Kramer for the SCC tissues, Dr
Jeffery S. Cox for the use of his freeze-drying facility, Dr
Richard Jordan for critically reading this manuscript, and
Stephen Ordway for editorial assistance.
4. Rubin MA. Use of laser capture microdissection, cDNA
microarrays, and tissue microarrays in advancing our under-
standing of prostate cancer. J Pathol 2001;195:80-6.
5. Emmert-Buck MR, Bonner RF, Smith PD, Chuaqui RF, Zhuang
Z, Goldstein SR, et al. Laser capture microdissection. Science
1996;274:998-1001.
6. Bonner RF, Emmert-Buck M, Cole K, Pohida T, Chuaqui R,
Goldstein S, et al. Laser capture microdissection: molecular
analysis of tissue. Science 1997;278:1481-3.
7. Ramos DM, Chen BL, Boylen K, Stern M, Kramer RH,
Sheppard D, et al. Stromal fibroblasts influence oral squamous-
cell carcinoma cell interactions with tenascin-C. Int J Cancer
1997;72:369-76.
8. Simone NL, Bonner RF, Gillespie JW, Emmert-Buck MR,
Liotta LA. Laser-capture microdissection: opening the micro-
scopic frontier to molecular analysis. Trends Genet 1998;14:
272-6.
9. Gu MB, Choi SH, Kim SW. Some observations in freeze-drying
of recombinant bioluminescent Escherichia coli for toxicity
monitoring. J Biotechnol 2001;88:95-105.
10. Arap W, Pasqualini R, Ruoslahti E. Cancer treatment by targeted
drug delivery to tumor vasculature in a mouse model. Science
1998;279:377-80.
11. Pasqualini R, Ruoslahti E. Organ targeting in vivo using phage
display peptide libraries. Nature 1996;380:364-6.

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