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A Standard Protocol for the Production and Bioevaluation of Ethical
In Vivo Models of HPV-Negative Head and Neck Squamous Cell
Carcinoma
Patrizia Sarogni, Ana Katrina Mapanao, Sabrina Marchetti, Claudia Kusmic, and Valerio Voliani*
Cite This: https://doi.org/10.1021/acsptsci.1c00083
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ABSTRACT: Preclinical cancer research increasingly demands
sophisticated models for the development and translation of
efficient and safe cancer treatments to clinical practice. In this
regard, tumor-grafted chorioallantoic membrane (CAM) models
are biological platforms that account for the dynamic roles of the
tumor microenvironment and cancer physiopathology, allowing
straightforward investigations in agreement to the 3Rs concept
(the concept of reduction, refinement, and replacement of animal
models). CAM models are the next advanced model for tumor
biological explorations as well as for reliable assessment regarding
initial efficacy, toxicity, and systemic biokinetics of conventional
and emerging neoplasm treatment modalities. Here we report a standardized and optimized protocol for the production and
biocharacterization of human papillomavirus (HPV)-negative head and neck chick chorioallantoic membrane models from a
commercial cell line (SCC-25). Oral malignancies continue to have severe morbidity with less than 50% long-term survival despite
the advancement in the available therapies. Thus, there is a persisting demand for new management approaches to establish more
efficient strategies toward their treatment. Remarkably, the inclusion of CAM models in the preclinical research workflow is crucial
to ethically foster both the basic and translational oncological research on oral malignancies as well as for the advancement of
efficient cancer treatment approaches.
KEYWORDS: chick chorioallantoic membrane, head/neck, cancer, alternative models, 3Rs principle, ethics
Head and neck squamous cell carcinomas (HNSCCs)
represent a wide class of epithelial neoplasms localized in
the oral and nasal cavities, paranasal sinuses, salivary glands,
pharynx, and larynx and whose molecular mechanisms involved
in the progression of the disease are still to be completely
clarified.
1,2
One of the leading causes of the development of
HNSCCs is the long-term consumption of tobacco and
alcohol.
3
Another associated risk factor for the onset of
HNSCCs is human papillomavirus (HPV) infection.
4
The
presence of HPV usually affects the prognosis of the disease,
with more favorable outcomes for the patients.
4,5
The general
workup for staging and diagnosis of squamous cell carcinoma
of the oral cavity, larynx, oropharynx, and hypopharynx
includes physical examinations, medical history, blood test,
MRI/CT imaging, and biopsy under local anesthesia.
4
These
features together with the evaluations on the genomic
alterations or gene expression profile are important for the
pathological staging and prognosis and to determine the type
of treatment. In general, the usual management strategies for
oral cavity, laryngeal, oropharyngeal, and hypopharyngeal
cancers are different between locally advanced tumors and
early stage tumors.
6
Indeed, according with the most recent
Clinical Practice Guidelines in Oncology (e.g., NCCN), the
standard treatments in early stage disease, consisting of
conservative surgery or radiotherapy, give similar locoregional
control. Standard options for locally advanced HNSCC are
either surgery plus adjuvant chemo/radiotherapy or primary
chemoradiotherapy alone, even if the best treatment regime is
chosen on a case-by-case analysis.
4,7
In the field of prognostic
estimation, it appears that cases of HNSCC that survive
beyond 5 years after the initial diagnosis show decreased
overall survival when compared to noncancer subjects of the
same age.
8
In general, stage I cases had improved survival
compared to stage II−IV, where no particular difference was
proved in long-term survival for cases alive 5 years after
diagnosis. However, site, stage, smoking, and cardiovascular
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disease are significant factors determinant of mortality.
8
Nevertheless, oral malignancies, including tongue cancer,
continue to have severe morbidity with less than 50% long-
term survival despite the advancement in the available and
emerging therapies.
4,5,9,10
In this regard, understanding the
biological processes at the basis of oral malignancies and the
development of new therapeutic strategies to improve the
survival rate of patients while preserving the structure and
function of the involved organs are crucial topics for their
management.
11
In this context, in vivo models are pivotal to
foster the advancements in oncology by bridging the gap
between preclinical investigations and human clinical trial on
conventional and emerging therapeutic approaches as well as
to understand tumor cell behaviors in a physiological
environment.
19
The most widely employed in vivo models
are murine, and they have been crucial to establish most of the
current models in pediatric oncology and to develop some
drugs, among which topotecan and irinotecan.
7
Moreover,
murine models are particularly relevant for absorption−
distribution−metabolism−excretion−toxicity (ADMET) in-
vestigations.
12−14
However, genetically immunocompromised
murine models have very high costs of maintenance, and the
tumor engraftment may require up to 4 months. Engraftment
failure can be high and cannot be usually determined before
some months post-implantation, and their employment is
increasingly discouraged by following the 3Rs concept (the
concept of reduction, refinement, and replacement of animal
models) and the European Parliament Directive 2010/63/
EU.
15,16
Among other biological models, the chick chorioal-
lantoic membrane (CAM) is one of the most attractive and
ethical in vivo models that jointly combines reliability,
medium-/high-throughput screenings, and easy handling
during imaging/treatment evaluations.
17−19
Notably, the
absence of a mature immune system of the embryo during
the early developmental stage reduces the risk of tumor
rejection after implantation.
19
Thus, CAM models can develop
visible solid tumors within 4−5 days after engraftment, in
comparison to the approximately 3−6 weeks of murine
models.
7
Besides the usually high rate of success for tumor
grafting, other major advantages of CAM models are the easy
daily inspection of the tumor site and their flexibility of
employment. Moreover, CAM models are ethical models in
agreement with the 3Rs concept because the chick embryo
does not develop pain perception before the 17th day of
incubation.
19
Indeed, investigations in CAM models usually do
not require permissions or approval from ethics committees.
19
Despite some difficulties in the application of CAM models to
long-term investigations, their highly vascularized membrane
together with the immature immune response allowed the low
rejection rate grafting of several tissues and the study of various
neoplasms, including osteosarcoma, glioblastoma, pancreatic
carcinoma, and colon carcinoma.
20−23
Several variable tumor
engraftment methods for CAM model production have been
reported, yet these studies focus on the tumor biology or the
evaluation of a treatment avoiding reporting a standard
protocol for the production of the models.
24,25
In general, in
the literature there is a serious lack of work regarding the
standard production of CAM models. Thus, a detailed step-by-
step procedure for the reliable CAM tumor model fabrication
together with the standard biocharacterization is required to
serve as guide for researchers interested to advance the field.
In order to address this demand, we report a standardized
protocol for the composition as well as the cascade assays for
the characterization of a commercial HPV-negative head and
neck cell line (SCC-25) grafted on chick chorioallantoic
membrane of fertilized Leghorn chicken eggs.
■MATERIALS
Reagents and Consumables.
•Fertilized red or white Leghorn chicken eggs
•SCC-25 squamous cell carcinoma cell line (ATCC,
catalog number CRL-1628)
•DMEM/Ham’s F12 1:1 medium (DMEM/F12 Gibco,
21041025)
•Fetal bovine serum, qualified, heat-inactivated (FBS,
Thermo Fisher Scientific, 10500064)
•L-Glutamine (Thermo Fisher Scientific, A2916801)
•Hydrocortisone (Sigma-Aldrich, H0888)
•Penicillin−streptomycin (Pen/Strep 100X, 5,000 U/
mL) (Thermo Fisher Scientific, 15070063)
•Phosphate-buffered saline without calcium and magne-
sium (PBS, Sigma-Aldrich, D8537)
•Trypsin-EDTA (0.5%), phenol red (Thermo Fisher
Scientific, 25300054)
•Serological pipette, pipette tips, microcentrifuge tubes,
conical tubes, and flasks
•TC Dish150, Standard (83.3903, SARSTEDT)
•Matrigel Matrix (Corning, ref 354234)
•Sterile water
•Fixative solution, i.e., 4% paraformaldehyde (PFA) in
PBS
•RNA extraction kit, Nucleospin RNA plus (740984.50
MACHEREY-NAGEL)
•cDNA synthesis kit, iScript cDNA Synthesis (1708891
BIORAD)
•iTaqUniversal SYBRGreen Supermix (1725121 BIO-
RAD)
•RIPA buffer (Pierce 89901)
•Protease Inhibitors Cockatil Tablets (04693116001
Roche)
•Bradford Reagent (B6916 Sigma-Aldrich)
•Albumin Standard (23209 Thermo scientific)
•Nitrocellulose membrane, TransBlot Turbo Midi-size
nitrocellulose (1620167 BIORAD)
•anti-TFRC primary antibody (SAB4200398 Sigma-
Aldrich)
•Goat anti-rabbit IgG (H+L)-HRP-conjugated secondary
antibody, (170−6515 BIORAD)
•Clarity Western ECL substrate (1705061 BIORAD)
•Paraffin wax (melting point 56 °C)
•Ethanol (70, 80, 95, and 100% alcohol)
•Xylene
•Mayer’s hematoxylin solution
•Eosin Y aqueous solution 1%
•Permount mounting medium
Equipment and Tools.
•Cell counter (Invitrogen Countess cell counter)
•Optical microscope
•Egg incubator, 37.5 °C/99.5 °F, 60% humidity, FIEM
MG 140/200
•Tilting egg racks
•Sterile dissection scissors and tweezers
•Soft tissue paper or cotton swab
•Refrigerator or cold room at 4 °C
•Analytical balance
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B
•Ruler
•Adhesive tapes, preferably Scotch magic tape
•Portable digital microscope, Dino-Lite AM7915MZT
(or any camera-associated microscope)
•DinoCapture 2.0 Software
•Rotary microtome
•Forced ventilation histology oven
•Paraffin-embedding station
•Light microscope equipped with RGB video camera
■METHODS
Experimental Outline. The establishment of the exper-
imental design is the first important step to define a complete
outline of the assay procedure. For the optimization of the
CAM assay employing SCC-25 cells, the schedules of egg
incubation and subsequent experimentations were based on
the proposed scheme by Kleibeuker et al. (Figure 1).
26
In
general, the start of the incubation corresponds to the
embryonic day of development 0 (EDD0). Starting from this
moment, under appropriate environmental conditions, the
fertilized eggs begin their embryonic growth. It is important to
take into account that the biological window available to
perform this assay must not exceed the 17 days of incubation
to prevent the hatching of the eggs and avoid ethical
restrictions.
19
Cleaning the Working Area and Eggs (Start of
Incubation). It is highly recommended to work under sterile
conditions and to adequately clean the working area with 70%
ethanol and/or bleach to avoid any type of microorganism
contamination. Carefully clean the shell of each egg with soft
tissue paper soaked in distilled water before fitting them into
the tray.
27
Place the eggs horizontally next to each other and
insert a steel spring in the ends of the columns to secure the
eggs while the racks tilt (Figure 2A).
28
Make sure that the
temperature and humidity of the incubator reach the
appropriate parameters before starting the incubation (37.5
°C, ∼47% humidity).
29
Puncturing. Take the eggs out after 3 days of incubation
(EDD3) and put them in upright position so that the air sac
will translocate to the top of the eggs.
30
Gently scratch with
tweezers the tip of each egg until the shell becomes fragile and
breakable, finally making a small hole. Seal the hole with an
adhesive tape to prevent dehydration and infection, and put
the eggs back in the incubator in the stationary mode (no
tilting).
Tumor Grafting. Selection of Eggs. At EDD6, the
fertilized eggs must be accurately selected before proceeding
with the grafting of the tumor cell suspension on the CAM.
Remove the eggshell around the small hole to create a small
window of about 1 cm2.
31
Check the fertilization and discard
the unfertilized eggs (Figure 2C). Label the eggs with the
appropriate information (e.g., egg number, cell density, name
of the cell line, and treatment conditions) to avoid confusion.
Reseal the window, and put the eggs back in the incubator.
Preparation of Tumor Cell Suspension. Harvest the SCC-
25 cells, and collect them in one 50 mL tube.
32
Spin down the
cells, and discard the supernatant. Resuspend the pellet in fresh
cell culture medium. Count the cells, and adjust the
concentration in order to have at least 2 ×106cells per
egg.
33
Spin down the cells again, and discard the supernatant.
Resuspend the pellet in a mixture of Matrigel/medium without
FBS (in ratio 1:1) such that each egg will be dispensed with 25
μL of the cell suspension.
34
Grafting. Open the sealed window. Roll a soft tissue paper
and gently poke a small vein within the CAM region until it
starts to bleed (Figure 2D).
35
Pipette 25 μL of the cell
suspension on top of the bleeding blood vessel. Reseal the
window, and put the eggs in the incubator (Figure 2E). Always
make sure that the humidifier and the tank are filled with water
and that the incubator is still maintaining the right conditions
of temperature and humidity.
Figure 1. Overview of the CAM schedule from EDD0 to EDD17. In general, chick embryo incubation is marked as embryonic day of development
0 (EDD0). The puncturing day (EDD3) allows the translocation of the natural air sac to the top of the egg. Grafting of 2 ×106SCC-25 cells
(EDD6) enables the generation of a solid visible tumor at 4 days post-grafting (EDD10). The topical treatment is applied on EDD10, and the
tumor mass is monitored until EDD17, the last day of incubation (harvesting).
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C
Apply Treatment (EDD10). A reference concentration of
your drug/chemotherapeutic compound to be tested is
recommended.
36
The amount of the treatment solution to
be prepared will depend on the number of tumor-grafted eggs
to be treated, while the frequency of the treatment will depend
on your own schedule and experimental objectives. To
proceed, take the eggs out from the incubator, and mark
each egg for the corresponding treatment to avoid confusion.
For our experiments, each egg is topically administered with
chemotherapeutic drugs/test compounds suspended in 30 μL
of solution (serum-free medium).
37
Tumor Growth Monitoring (EDD10−EDD17). Place a
ruler under the DinoLite camera and calibrate the software
before starting to acquire images of the tumors. Although
constantly noting the calibration by using the ruler can help in
obtaining consistent and accurate measurements, it is also
advised to take photographs at the same magnification to
better visualize the changes in tumor dimensions. The
complementary DinoLite software (DinoCapture 2.0) comes
with length measurement tools. Measure the tumor sizes, in
which the longer and shorter measurements are denoted as the
length (L) and width (W), respectively (Figure 2F). From
these measurements, the tumor volume is derived using a
modified ellipsoid formula: 0.5(L×(W2)).
38,22
Tumor Harvesting (EDD17). On EDD17, take the eggs
out of the incubator and place them in the cold room for at
least 2 h to restrict the movements of the chick embryo.
Thoroughly clean the dissection area, tweezers, and scissors
with 70% ethanol and bleach. Prepare wash containers with
ethanol and PBS for rinsing the tools to avoid cross-
contamination among different tumors. Open the sealed
window, and remove some eggshell around to make the
tumor more accessible for cutting. Carefully lift the membrane
with tweezers. Cut the tumor, and place it in a Petri dish with
PBS. Take a photograph of the tumor, and place it in an empty
microcentrifuge tube, and weigh it (Figure 2G).
39
Store the
harvested tumor samples directly in −80 °C or in formalin
fixing solution for further analysis.
■DOWNSTREAM ASSAYS
Quantitative Real Time-PCR. To extract the total RNA
from the SCC-25 cell line, we used Nucleospin RNA plus Kit
(740984.50 MACHEREY-NAGEL) following the manufac-
turer’s instruction. Briefly, the harvested tissue is minced in
small pieces using a plastic pestle. The extracted RNA can be
used immediately or stored at −80 °C. The quality control of
RNA through agarose gel electrophoresis will avoid the
likelihood of final problem solving, such as in cDNA reverse
transcription and amplification of the gene target. For cDNA
synthesis, 500 ng of RNA was reverse-transcribed with iScript
cDNA Synthesis Kit (1708891 BIORAD). Dilute 500 ng of the
total cDNA 1:10 in nuclease-free water in order to get 50 ng as
final amount before using it for the PCR reaction. Quantitative
real-time PCR is carried out using iTaqUniversal SYBRGreen
Supermix (1725121 BIORAD). To prepare the PCR reaction
mix in a final volume of 20 μL, use 1−2μL of the diluted
cDNA with around 5−10 ng of cDNA template. Use specific
primers for your gene of interest and primers for a
housekeeping gene. One of the commonly utilized house-
keeping genes, which we also use, is GAPDH. All the samples
are prepared in triplicate. The amplification curves are
visualized by SYBR Green Analysis on Applied Biosystem
Instrument (7300). The recommended thermal cycling for the
amplification is as follows: 95 °C for 10 min, 40 cycles at 95 °C
15 s, 64 °C for 30 s, and 72 °C for 30 s. The 2−ΔΔCT method is
used to calculate the relative expression level.
40
Western Blotting. Cells pellets are resuspended in RIPA
buffer (Pierce 89901) supplemented with protease inhibitors
and mechanically minced using a 200 μL pipet tip. The lysates
Figure 2. Images of the main steps of the CAM protocol. (A) Eggs are
horizontally placed in the tilting trays before the incubation starts. (B)
A light source placed on top/behind the egg allows identification of
the location of the air chamber and the existence of a vascular network
(Top, infertile egg; bottom, fertile egg). (C) An unfertilized egg is
associated with the absence of the embryo and of the vascular
network. (D) Distinction between artery (arrow) and vein (asterisk).
Arteries are thicker and darker compared to veins.
41
(E) Image
showing the grafting procedure of SCC-25 cancer cells onto the
bleeding blood vessel. (F) Tumor volume measurement determined
by means of width and length. The length is associated with the
longest diameter of the tumor mass. (G) Harvesting procedure of the
tumor at EDD17. The CAM membrane is gently lifted with tweezers
and the tumor is cut with scissors.
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D
are then incubated for 30 min in ice, and the supernatants are
collected after centrifugation for 30 min at 14 000 rpm. The
protein concentration in the lysates is determined through the
Bradford assay, using a standard calibration curve method
prepared with bovine serum albumin (BSA) of known
concentrations (2000 μg/mL). The absorbance values at 595
nm of the samples and the standards are noted. The formula y
=mx +qis used to derive the protein concentration. Then,
30−50 μg of the total protein was separated via SDS-PAGE.
Upon transferring the samples from the gel to a nitrocellulose
membrane, the proteins are treated with a blocking solution
(TBS 1×5% powdered milk) for 1 h at room temperature. For
the overnight primary antibody incubation at 4 °C, we used
anti-TFRC primary antibody (SAB4200398 Sigma-Aldrich).
The membrane was washed thrice with TBS 1×−0.1%
Tween20, and the horseradish peroxidase (HRP)-conjugated
secondary antibody was then added and incubated for 1 h at
room temperature. Finally, after further washing in TBS 1×−
0.1% Tween20, the bands are visualized through chemilumi-
nescence using an enhanced chemiluminescence (ECL) kit
(1705061 Biorad) and Image Quant LAS 4000 System.
Hematoxylin and Eosin (H&E) Staining. Tumor
samples, fixed in 4% paraformaldehyde for 24−48 h, were
rinsed in running tap water for 10−15 min. Then, dehydrate
samples through increasing alcohol series, followed by three
changes of 100% alcohol applied for 5 min each. The tissues
were cleared in xylene for 12 min and then immersed in
paraffin wax for 10 min, followed by other two paraffin
changes, 5 min each, and finally embedded in paraffin blocks.
Serial sections of 5−6μm were cut with a microtome, placed
on slides, and heated overnight at 40 °C in forced ventilation
histology oven. The sections were cleared from paraffin by two
changes in xylene, 6 min each. The tissue was hydrated
through decreasing ethanol series and rinsed in distilled water
for at least 5 min.
Slices were stained with Mayer’s hematoxylin solution for 5
min, followed by a 10 min rinse in running tap water, and
finally stained in eosin Y aqueous solution for 2 min. Slides
were rinsed in distilled water, heated at 40 °C for 40 min,
dipped twice in xylene, and a coverslip was added placing a
drop of Permount mounting medium.
Histological images (40×, 100×, 200×, and 400×) are
acquired by light microscope (Olympus BX43, Japan) and
digitized using a RGB video camera (Olympus DP 20, Japan).
■ANTICIPATED RESULTS
ThereliablegenerationofasolidtumoronCAMis
fundamental for both the comprehension of in vivo cancer
cell behaviors and the efficacy/toxicity evaluation of emerging
and conventional therapeutic strategies. Imaging of tumors
provides detailed information about the quality and the
presence/absence of blood vessel across the tumor itself; an
important parameter to monitor during the time frame of the
experiment and/or after the therapeutic treatments.
42
Imaging
also provides a practical method to identify the volume of the
tumor mass without interfering with its spatial organization
and allows for distinguishing the tumor from artifacts caused
by the aggregation of the Matrigel solution. The primary goals
in the production of CAM tumor models include the
achievement of a high embryo survival rate and visible tumor
grafting that allow the topical application of therapeutics. Our
optimized protocol allows the development of solid vascular-
ized SCC-25 tumor with high efficiency (∼80%) and with a
volume of 5−20 mm3at EDD10 (Figure 3A). It is important
to notice that the assessment of tumor size following the
harvesting can be affected by the wrinkling of the membrane
Figure 3. Characterization of harvested SCC25 tumors. (A) Representative image of SCC-25 solid tumor grown onto the CAM. The arrows
indicate the blood vessels across the tumor mass. (B) Example of solid and vascularized tumor harvested at EDD17. (C) Western blotting analysis
depicting the expression of the TfR marker in SCC-25-tumor-derived cells. (D) H&E staining of SCC-25 tumor-derived cells showing the tissue
structure and cells distribution (TC = tumor cells; S = stroma; scale bar = 20 μm). (E) Real-time PCR measurement of VEGF-A mRNA expression
levels in SCC-25 cancer cells compared to HBEpc bronchial cell line.
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E
after 2 h at 4 °C. However, the size of the excised tumors
usually significantly correlates with their in vivo volume.
Measuring the weight of the excised tumors is a facile and
useful end-point evaluation that provides additional informa-
tion on the aggressiveness and ability of cancer cells to grow
and form solid tumors (Figure 3B). It is worth remembering
that the identification and introduction of new molecular
pathways involved in the neoplastic transition has rapidly
expanded, considerably advancing the diagnostic techniques.
43
In fact, the detection of specific biomolecular tumor markers
represents a significant diagnostic screening approach that
allows differentiation between cancer cells and the surrounding
cells. Among the number of molecular mechanisms already
identified in neoplasms, the expression of transferrin receptor
(TfR) appears to be compromised in HNSCCs, leading to an
overexpression of the receptor and therefore constituting a
promising tumor marker.
44,45
In this regard, to effectively
prove the human origin of the harvested tissue, Western blot
analysis has been carried out using an antihuman TfR
antibody. The results demonstrate the presence of the TfR
protein maker in the harvested tumor samples, confirming the
significance of this approach to determinate the human origin
of the grafted tumor (Figure 3C).
H&E staining is another pivotal end-point assay that mainly
provides qualitative information and a general overview on the
structure of the tissues.
46
Remarkably, this approach enables
visualization of cellular morphology and the distribution
pattern of distinct cells, as well as their density and consistency.
The H&E staining of the collected SCC-25 tumor specimen
showed a well-organized, preserved, and homogeneous
structure, clearly identifiable from the surrounding stroma
and the membrane border (Figure 3D).
Since genetic alterations are often events upstream of cell
metabolism dysfunction, advances in the assessment of gene
expression profile may be useful for the prognosis of various
type of cancers, hence offering a wide overview about the gene
interaction network in the development of the disease. In fact,
mutations that lead to the alteration of gene expression level of
cytokines, enzymes, and growth factors are the main
responsible of tumorigenesis.
47
The deregulation of the
vascular endothelial growth factor-A (VEGF-A) was observed
to be among the tumor promoting factors in SCC-25 cells.
Indeed, VEGF-A is relatively overexpressed in SCC-25 cells
compared to the level in normal cells (Figure 3E) and
constitute a promising molecular therapeutic target for head
and neck tumors. Overall, these findings aim to emphasize the
use of the chick embryo as an in vivo model for the screening of
anticancer compounds.
■SUMMARY
HNSCCs represent an aggressive class of neoplasms with a
worldwide high incidence due to two main risk factors:
tobacco/alcohol consumption and HPV infection.
48
The
translation of efficient therapeutic strategies in oncology
requires a deep understanding beyond the physiopathology
of neoplasms as well as ethical and reliable in vivo models that
provide an analogous environment for an accurate exploration
of the behavior of cancer tissues and their response to the
therapeutic approaches. Due to its highly vascularized
environment and immature immune responses, the CAM
model provides an optimal environment for the grafting of
cancer cell lines and patient-derived cancer cells.
49
Here, we have presented a standardized and optimized
protocol for the medium-/high-throughput production of solid
tumors using the commercial HPV-negative head and neck cell
line SCC-25. Each step of the protocol requires basic biological
practice and experience. In general, the high survival rate of the
embryos and tumor take rate are among the essential criteria
considered for the efficiency of the protocol. Additionally, a
detailed outline and explanation of the steps and further
remarks have been included to provide a suitable guide for the
generation of tumor grafts and subsequent imaging and
biomolecular assays. The reported step-by-step method allows
the establishment of a feasible in vivo model that can provide
insights on the biological processes at the basis of oral
malignancies and the development of new therapeutic
strategies.
50
The CAM model may push oncological research
toward a more rapid evaluation and efficient screening/
selection of conventional and emerging antitumor treatments.
■AUTHOR INFORMATION
Corresponding Author
Valerio Voliani −Center for Nanotechnology Innovation@
NEST, Istituto Italiano di Tecnologia, Pisa 56126, Italy;
orcid.org/0000-0003-1311-3349;
Email: valerio.voliani@iit.it
Authors
Patrizia Sarogni −Center for Nanotechnology Innovation@
NEST, Istituto Italiano di Tecnologia, Pisa 56126, Italy
Ana Katrina Mapanao −Center for Nanotechnology
Innovation@NEST, Istituto Italiano di Tecnologia, Pisa
56126, Italy; NEST-Scuola Normale Superiore, Pisa 56126,
Italy
Sabrina Marchetti −Institute of Clinical Physiology, CNR,
Pisa 56100, Italy
Claudia Kusmic −Institute of Clinical Physiology, CNR, Pisa
56100, Italy
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsptsci.1c00083
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
This work is supported by Associazione Italiana per la Ricerca
sul Cancro (AIRC) under MFAG 2017, ID 19852 project
(P.I.: V.V.). Figure 1 and the graphical abstract have been
created with BioRender.com.
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(27) Kunz et al. demonstrated that the application of 70% ethanol or
other disinfectants on the shell considerably reduces the viability of
the embryo by approximately 30%, thereby affecting the overall
execution of the experiment.
20
(28) The tilting racks are usually provided upon the purchase of the
incubator. They have a metal pin that fits into a small hole in the back
of the incubator, hence allowing the automatic movement. If no tilting
racks are available, then the trays can be manually moved through
180°at least 2 or 3 times per day.
(29) Once all the eggs are inside and incubation starts, it is
recommend to avoid frequent opening of the incubator in order to
keep the temperature and humidity constantly on the set parameter
and not interfere with the growth of the embryo. The percentage of
the unfertilized eggs may interfere with the final outcome of the
experiment, and for this reason, it is strongly recommended to start
with a large number of eggs to obtain consistent result, e.g., 10% more
than expected eggs for the experiment. It should also take in
consideration that the fertilization rate of the eggs can be seasonal
depending on the supplier.
(30) Placing a bright light source on top/behind the egg
(“candling”) allows you to see through the shell. The air chamber
should be located in the blunt end of the egg. The incubated eggs can
be also candled to check for fertilization. While the fertilization of
white eggs can be checked at the third day of incubation, the brown-
shelled eggs may require a few more days to check this parameter
(Figure 2B). The presence of vessels is an indication of fertilized egg.
(31) The small window should be carefully opened to avoid shell
debris falling onto the membrane. If this happens, then try to carefully
remove the debris with sterile tweezers, without damaging the
membrane, to prevent infections or microorganism contamination.
(32) SCC-25 cells are maintained in a complete growth medium
composed of a 1:1 mixture of Dulbecco’s modified Eagle’s medium
and Ham’s F12 medium supplemented with 10% of fetal bovine
serum (FBS), 4 mM L-glutamine, 1mM sodium pyruvate, 100 U/mL
penicillin, and 100 mg/mL streptomycin. The medium is also
supplemented with 400 ng/mL hydrocortisone. It should be noted
that SCC-25 is a very adherent cell line. Thus, a 5 min incubation in
trypsin may not be sufficient to completely detach the cells from the
culture dish. After the medium is removed, it is recommended to first
wash the cells with PBS and, then, with a small amount of trypsin.
After discarding, add trypsin again and incubate the cells for 5−10
min to harvest a high and consistent number of cells. Knowing the
doubling time of your cells may be useful for setting up the
experimental design. For SCC-25, the doubling time was observed to
be approximately 45 hours. It is also suggested to use 150 mm plates
to promote an extensive cell growth and to lower the frequency of
splitting the cells.
(33) The final cell number per egg was empirically determined. It
has been observed that 2 ×106cells for SCC-25 is the minimum
amount needed to obtain solid tumor formation by EDD 10 (i.e., 4
days post-grafting and assigned EDD in which treatments are initially
administered).
(34) Matrigel is a protein mixture that resembles the extracellular
environment required to have a conducive substrate for cell growth.
Moreover, it is a dense mixture and keeps the cells together once
deposited on the membrane. The Matrigel solution is stored at
−20°C. It needs to be thawed overnight at 4°C before the grafting
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experiment. It is recommended to prepare Matrigel aliquots to keep
the stocks in proper storage temperature and avoid repeated freeze−
thaw cycles. While working with Matrigel, it should be kept constantly
on ice to prevent polymerization while preparing the working solution
(12.5μL Matrigel + 12.5μL medium without FBS per egg) or
dispensing the cells on the membrane. Importantly, the tumor grafting
efficiency at EDD 10 is only 20% if the SCC-25 cells deposited on the
CAM are suspended in the medium alone.
(35) The veins and arteries of the CAM are easily distinguished from
each other by the different blood color and vessel motility.
41
The
arteries are darker, and their size is larger compared to the veins. Also,
the movement of the arteries is more active, and their walls are
thicker, making them difficult to rupture during cell grafting.
(36) It usually takes 4 days post-grafting for the SCC-25 cells to
grow and form a solid visible tumor on the CAM. This timeframe may
change for other cell lines. It is recommended to have at least 8−9
eggs per treatment condition in order to have consistent statistics as
well as to employ at least 3 eggs as a control group without any
treatment.
(37) The treatment solution must be freshly prepared just before its
application. It has been observed that 30 μL of solution is enough to
adequately cover the tumor mass. Do not touch the tumor with the
tip while applying the treatment in order to avoid tissue damage.
(38) The tumor measurements from EDD10 to EDD14 are taken
from a superficial angle. Indeed, their volume is calculated by
considering the width and length of the visible tumor estimated from
the top of the CAM. It should be considered that some neoplasms
may grow below or above the membrane. Image acquisition can be
time consuming, especially when handling a lot of samples. It is
recommended to always keep the same magnification for all the
collected images. Any magnification changes require a software
recalibration. The light and embryo movements may interfere with
the acquisition of good quality images. It is also important to adjust
the light and polarization of the microscope to remove as much light
reflection as possible on the membrane in order to collect high-quality
images. Eggs should be taken out from the incubator in small batches
during visual analysis to prevent the frequent opening and closing of
the incubator, which can affect the settings.
(39) Considering that the appearance of the membranes and the
tumors can change after 2 hours at 4 °C due to the wrinkling of the
CAM, the visualization of the tumor can be difficult. In this regard,
the images from previous EDDs may be helpful to locate the position
of the tumor. While harvesting the tumor, cut as little of the
membrane as possible since it might interfere with the weight of the
tumor and other downstream assays such as RNA extraction and
analysis of gene expression profile, protein extraction and detection of
tumor marker, and qualitative information about the structure of the
tissue.
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