Supplemental Materials and Methods
Cell Lines. All cell lines were obtained and validated by the following sources. HPV status of
the following cell lines has been previously validated (1). HPV negative cell lines: UM-SCC1,
UM-SCC6, UM-SCC22B, and UM-SCC1483. HPV positive cell lines: UD-SCC2, UM-SCC47,
UPCI-SCC90, and 93-VU-147T.
Cell line
UM-SCC1
UM-SCC22B
UD-SCC2
UPCI-SCC90
93-VU-147T
(SCC147T)
UM-SCC47
UM-SCC4
UM-SCC6
UM-SCC11A
UM-SCC14A
UM-SCC38
UM-SCC104
UM-SCC1483
HN4
Sources
Culture condition
Dr. Thomas E. Carey, University of
Michigan (2)
DMEM with 4.5 g/dL glucose,
10% FBS, 1% hydrocortisone,
penicillin (100 units/mL),
streptomycin (100 mg/mL)
Dr. Thomas Carey, with permission of Dr.
Henning Bier, Technical University
Munich, Munich, Germany
Dr. Robert Ferris, University of Pittsburgh
Dr. Robert Ferris, with permission of Dr.
Hans Joenje, VU Medical Center,
Amsterdam, Netherlands
Dr. Thomas E. Carey, University of
Michigan (2)
DMEM with 4.5 g/dL glucose,
10% FBS, penicillin (100
units/mL), streptomycin (100
mg/mL)
Dr. Jennifer Grandis, University of
Pittsburgh
Dr. Ravi Salgia, University of Chicago
Clonogenic Survival Assay: Cells were plated in 6-well plates (100-500 cells/well) in medium
containing 20% serum. Cells were pre-treated with 0.25 uM of R428 or vehicle. Cells were
irradiated 24 hours later at the doses of 2 or 4 Gy. After 10 to 14 days, colonies were stained with
crystal violet and manually counted. Colonies consisting of more than 50 cells were counted and
the surviving fraction was calculated; 18 replicate wells were counted per treatment.
Cell Line Xenografts, PDXs, and Radiation Response. Established HNSCC cell lines UMSCC1, UM-SCC22B, UD-SCC2, UM-SCC47, UPCI-SCC90 and 93-VU-147T were used to
generate xenografts as previously described (1). Mice were housed in the Wisconsin Institute for
Medical Research (WIMR) Animal Care Facility, and all experiments were carried out in
accordance with an animal protocol approved by the University of Wisconsin. When tumor
volumes reached approximately 200 mm3, mice were stratified into control (vehicle) or radiation
treatment groups (n=12 mice/24 tumors per group) such that all groups contained a range of
similarly sized tumors. Radiation therapy was administered as four 2 Gy fractions over two
consecutive weeks using an X-rad 320 biological irradiator (Precision X-ray, Inc.). After
completing the treatment regimen tumor volume was assessed twice weekly with Vernier
calipers and calculated according to the equation V = (π/6) × (large diameter) × (small
diameter)2; tumors were measured until the majority of control tumors quadrupled in size. Tumor
growth curves were generated using Graphpad Prism v6.0d. The mean tumor volumes were
compared between the control and radiation arms at the final three time points using two-sample
t-tests with equal standard deviations. Tumors with significant p-values at all three time points
were deemed sensitive to radiation, while those with non-significant (NS) p-values were
considered resistant. *p<0.05, **p<0.01.
PDX establishment, TMA construction and IHC for AXL were performed as previously
described (3, 4). Six PDXs were evaluated for radiation response by implanting bilateral
posterior flank tumors into 16 female Hsd:athymic Nude-Foxn1nu mice (Harlan Laboratories).
When tumor volumes (calculated as described above) reached approximately 200 mm3, mice
were stratified into control and radiation treatment groups (n=8 mice/16 tumors per group).
Radiation therapy was administered as four 2 Gy fractions (UW-SCC6 and UW-SCC22) over
two consecutive weeks or five 2 Gy fractions over five consecutive days (UW-SCC30, UWSCC1, and UW-SCC36) using an X-rad 320 biological irradiator (Precision X-ray, Inc.). Tumor
growth was measured and plotted as above.
63 HNSCC patient cohort and TMA construction. A TMA was constructed from formalin-fixed
paraffin embedded HNSCC tumor biopsies. 1.0 mm core extractions of each tumor biopsy were
represented on the tissue microarray by duplicate cores. AXL staining was performed via IHC
using mAb185 (1:50) developed in the laboratory of Dr. Parkash Gill (Department of Medicine
and Pathology, University of Southern California, Los Angeles, CA). 20x photographs of AXL
staining were taken using Aperio software (Leica Microsystems Inc, Buffalo Grove, IL, USA).
Statistical Analysis for 63 HNSCC patient cohort. AXL scores 0 and 1+ were characterized as
“low”, while AXL scores 2+ and 3+ were characterized as “high” by pathologist M.W.L. A
multiple logistic regression model was fit to identify any patient characteristics associated with
AXL score. The results were presented as odds ratios with associated 95% confidence intervals.
Progression-free survival (PFS) in this cohort was determined by evaluating how many patients
in the low and high AXL groups experienced a recurrence of their disease or passed away. The
comparison of PFS between patients with high and low AXL scores was evaluated using the
Kaplan Meier method. Results were summarized using median survival times and hazard ratios,
and the statistical significance between high and low AXL scores was assessed using the logrank test. Only 57 out of 63 patients could be used for the PFS analysis due to missing data. All
analyses were performed using statistical procedures in the SAS/STAT software (version 9.3),
and a p-value less than 0.05 was considered statistically significant.
Fractional Product Method for Drug Combination Synergy. The nature of the interaction
between R428 or siAXL and the individual therapies of chemotherapy, cetuximab, or
radiation was evaluated via the fractional product method described by Chou and Talalay
(5-7). Briefly, this method utilizes the relative cell density following treatment with each
individual agent and calculates the expected (E) effect of combination therapy as a product
of the individual responses. The observed (O) effect is the relative cell density following
dual treatment. A ratio of the observed to expected (O:E) values was calculated and used to
estimate the synergy, additivity, or antagonism. A value less than 1 indicated synergism,
greater than 1 demonstrated antagonism, and 1 represented additivity.
References
1. Kimple RJ, Smith MA, Blitzer GC, Torres AD, Martin JA, Yang RZ, et al. Enhanced radiation
sensitivity in HPV-positive head and neck cancer. Cancer Res. 2013;73:4791-800.
2. Brenner JC, Graham MP, Kumar B, Saunders LM, Kupfer R, Lyons RH, et al. Genotyping of
73 UM-SCC head and neck squamous cell carcinoma cell lines. Head Neck. 2010;32:417-26.
3. Brand TM, Iida M, Stein AP, Corrigan KL, Braverman CM, Luthar N, et al. AXL Mediates
Resistance to Cetuximab Therapy. Cancer Res. 2014;74:5152-64.
4. Kimple RJ, Harari PM, Torres AD, Yang RZ, Soriano BJ, Yu M, et al. Development and
characterization of HPV-positive and HPV-negative head and neck squamous cell carcinoma
tumorgrafts. Clin Cancer Res. 2013;19:855-64.
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and antagonism in drug combination studies. Pharmacological reviews. 2006;58:621-81.
6. Chou TC. Drug Combination Studies and Their Synergy Quantification Using the ChouTalalay Method. Cancer Research. 2010;70:440-6.
7. Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of
multiple drugs or enzyme inhibitors. Advances in enzyme regulation. 1984;22:27-55.