In silico analysis of the expression of CA IX and CA XII in HNSCC
For CA IX and CA XII mRNA expression analysis in HNSCC patients the Genomics Analysis and Visualization platform (R2: Genomics analysis and visualization platform; http://r2.amc.nl) was used. The analysis was performed with the following datasets: GSE18674 which includes 22 human normal tissues and GSE42743 which includes 103 oral cavity tumors from Stanford University School of Medicine, Stanford Cancer Center (Standford, CA, USA). The correlation was assessed by one-way analysis of variance (ANOVA), through the R2 platform and presented in box plots.
Cell lines and culture conditions
The head and neck squamous carcinoma cell lines, FaDu and SCC-011, came from the American Type Culture Collection (ATCC, Manassas, VA) and Cellosaurus JHU-011 (CVCL_5986).
FaDu cells were cultured in Dulbecco Minimum Essential Medium (DMEM) whereas SCC-011 cells in the Roswell Park Memorial Institute (RPMI), supplemented with 10% fetal bovine serum (FBS) and 1% L-glutamine-penicillin–streptomycin and grown at 37 °C with 5% CO2.
All experiments were performed by growing FaDu and SCC-011 cells both in normoxic (21% O2) and in hypoxic conditions (1% O2). Hypoxia was attained in a modular incubator chamber (Stem Cell, Catalog #27,310). The chamber was flooded with the hypoxic gas mixture for 7 min and then sealed and stored in an incubator at 37 °C in 5% CO2. The normoxic control was stored in the same incubator for the same amount of time .
Cell viability assay
The viability of FaDu and SCC-011 cells (4 × 103 cells/well, 96-well plates), treated with SLC-0111 (100 µM) alone or in combination with different concentrations of Cis-Pt, was assessed with CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega BioSciences Inc., Fitchburg, WI, USA) using 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H tetrazolium (MTS), and according to the manufacturer’s instructions .
CA IX/XII inhibitor SLC-0111, developed in the laboratory of Professor Claudiu T Supuran (NEUROFARBA Department, University of Florence, Italy) and previously described , was used at 100 µM dose alone or in combination with Cis-Pt to evaluate a potential enhanced response of HNSCC cells to conventional chemotherapeutics.
Cell migration assay
Cell migration was performed as previously reported using 24-well Boyden chambers (Corning, NY) with inserts of polycarbonate membranes (8 µm pores). FaDu and SCC-011 cells (0.5 × 105 /well) were re-suspended in 100 µL of serum-free medium in the presence or absence of Cis-Pt and SLC-0111 (100 µM) and seeded in the upper chamber. After the addition of 10% FBS in the lower chamber as chemo-attractants, the trans-wells were put in a humidified incubator in 5% CO2 for 24 h at 37 °C. The non-migrated cells were removed with cotton swabs, whereas the cells that had migrated were visualized by staining the membrane with 0.1% crystal violet in 25% methanol.
10 random fields/filter were counted under a phase contrast microscope (Leica) and images were captured using a digital camera (Canon). All experiments were performed at least three times.
All the results are expressed as the percentage of migrating cells considering the vehicle control sample as 100% [18, 19].
Cell invasion assay
The invasion assay was performed using the Boyden chamber with membranes (8 µm pores) coated with 50 µL of diluted Matrigel (1:5 in PBS) (Corning, NY, USA). FaDu and SCC-011 cells (1 × 105 /100 µL serum-free medium per well) were harvested, suspended in serum free medium alone or containing Cis-Pt (1 µM) and SLC-0111 (100 µM) and placed in the top chamber. In the lower chamber, a medium containing 10% FBS was added and used as chemo-attractant. Cells were allowed 72 h to invade in a humidified incubator with 5% CO2 at 37 °C. To visualize and analyze invading cells, the same experimental procedure described above for cell migration assay was performed. All experiments were performed at least three times [20, 21].
Spheroid formation assay of HNSCC Cells
FaDu and SCC-011 cells (100 × 103 cell/well) were seeded in ultra-low attachment 6-multiwell-plates (Corning) and grown in spheroid medium containing serum-free DMEM supplemented with B27 (1X), bFGF (20 ng/mL) EGF (10 ng/mL). Cells were incubated at 37 °C with 5% CO2 for 7 days. Spheroid formation was analyzed under a phase-contrast microscopy and the size and number of formed spheroids were calculated using ImageJ .
3D invasion assay
FaDu and SCC-011-spheroids formed, as previously described, were embedded with a matrigel mixture in a ratio 1:1 with spheroid medium. After the spheroids were embedded, cell invasion out of the spheroids was monitored each 24 h. Representative images were acquired and sprouting length was calculated using ImageJ. All images were acquired with an inverted light microscope at 10 × magnification. Data is expressed as mean ± standard deviation of the relative invasive area of the spheroids after 48 h of hypoxia (1% O2) .
FaDu and SCC-011 cells (500 cells/well, six-well plates) were cultured with SLC-0111 (100 µM) alone or in combination with Cis-Pt (1 µM) at 37° for 14 days. After washes with DPBS, cells were fixed and stained with 0.1% crystal violet in 25% methanol. Following 30 min at RT, culture dishes were washed with DPBS and colonies were photographed. 1% Sodium Dodecyl Sulphate was added on the cells perfectly washed, in order to induce crystal violet dissolution. Absorbance was recorded at 490 nm by a 96-well-plate ELISA reader .
Cell lysate preparation and western blot analysis
An equal amount of proteins from cells were separated by 4–12% SDS-PAGE and were transferred to a nitrocellulose membrane. Blots were blocked for 1 h with 5% non-fat dry milk and then incubated over night with the following primary antibodies: HIF-1α (BD, Biosciences), CA IX (R&D), CA XII (Santa Cruz), anti-SOX-2 and anti Nanog (CST-9093; Cell Signaling Technology Inc), anti-N-cadherin, anti-E-Cadherin, anti-β-catenin (CST-9782; Cell Signaling Technology Inc), anti-Pro-Caspase-3, anti-cleaved-caspase-3, anti-PARP, anti-cleaved-PARP (CST-9915; Cell Signaling Technology Inc), anti-Stat3 (CST-9139), anti-pStat3 (CST-9138), anti-AKT (CST-9272), anti-pAKT (CST-9271), anti-ERK (CST-9102), anti-pERK (CST-9101), MMP-2 (CST-33437), anti-GADPH (Sigma-G8795), anti-Tubulin (Santa Cruz) and anti-Actin (Sigma-A4700). After washing with 0.1% Tween-20 in PBS, the filters were incubated with their respective secondary antibodies for 1 h and analyzed using the ECL system. Densitometric analyses were performed on at least two different expositions to assure the linearity of each acquisition using ImageJ software (v1.46r) [25, 26].
All experimental procedures complied with the European Communities Council directives (2010/63/EU) and national regulations (D.L. 116/92) and were performed in accordance with National Institutes of Health (NIH) recommendations. The present study was approved by the Italian Ministry of Health (authorization number 932/2018-PR). All procedures were performed according to FELASA guidelines for welfare of experimental animals and all imaging acquisition, as well as intravenous injections of fluorescent probes and orthotopic implantations, were performed under general anesthesia with isoflurane (2%) in oxygen (0.8 Lt/min). Each of the imaging workstations used has a heating system to maintain body temperature during the exams, and a system for physiological monitoring (rectal temperature, heart and respiratory rates). All efforts were made to minimize animal suffering and the number of animals necessary to produce reliable results.
FOXN1NU nude mice were subcutaneously injected in the right flank with 2 × 106 FaDu cells, resuspended in 0.1 ml of 1:1 mix of physiological saline and Matrigel. Once tumors became palpable (established), approximately 50 mm3 [volume = 0.5 × long diameter × (short diameter)2] , nude mice were randomized into 4 treatment groups (3 mice for each group): i) Cis-Pt – treated with i.p. injection of 3 mg/kg of Cis-Pt, in a total volume of 50 µL, every other day for 3 weeks; ii) SLC-0111 treated with per os administration, via oral gavage, of SLC at 100 mg/kg in a total volume of 100 µL, once per day, five days per week for 3 weeks, the oral formulation of SLC-0111 consisted of 55.6% PEG400/11.1% ethanol/33.3% water ; iii) combined therapy with Cis-Pt and SLC-0111 at the aforementioned dosages and administration schemes; iv) vehicle group receiving 100 µL of phosphate buffered saline i.p. every other day for 3 weeks. Mice were checked and weighted twice per week, and tumor mass were measured with a manual caliper during these operations. In order to develop an HNSCC orthotopic model 2 × 106 FaDu cells, resuspended in 0.1 ml of 1:1 mix of physiological saline and Matrigel, was gently injected in the left lingual muscular belly with the animal under general anesthesia. For the intravenous injection of fluorescent probes Annexin Vivo 750 and ProSense 750, animals were intravenously catheterized in one lateral tail vein of mice maintained under isofluorane anesthesia, using a catheter mounting a 30G needle. Then 2 nmoles of the probe were slowly injected and the catheter was further flushed with 10–20 µL of sterile saline. At the end of imaging studies mice were euthanized. Treatment schedule is schematized in Fig. 7A.
All mice were maintained on a diet with a purified, alfalfa-free rodent chow for 15 days before fluorescence imaging to minimize fluorescence in the gut. Near-infrared fluorescent molecular imaging was performed with Annexin Vivo 750 and ProSense 750 using FMT4000 (Perkin-Elmer Inc., Waltham, MA, USA). 2 nmol of Annexin Vivo 750 were administrated to each HNSCC s.c. xenografts and FRI was used to study in vivo xenografts accumulation of after 2 and 24 h, and ex vivo in the surgically harvested primary tumor mass. 2 nmol of Prosense 750 were administrated to each HNSCC orthotopic xenografts and FMT of head, neck and thorax was used to study accumulation in the orthotopic tumor, neck lymph nodes and lungs. The tongue including the orthotopic tumor and the before mentioned organs where surgically harvested and studied with FRI. Mice were placed in dorsal recumbency to study the orthotopic tumor, neck lymph nodes and lungs, in vivo, and in lateral recumbency for xenograft studies. At the end of the imaging acquisition mice were euthanized while under anesthesia and the aforementioned organs were harvested for ex vivo evaluation. Images were reconstructed with FMT system software (TrueQuantTM v4.0) from PerkinElmer (Waltham, MA). For FRI an elliptical region of interest (ROI) was designed over the xenograft excluding as much as possible of surrounding normal tissues, the total counts/energy were recorded for each animal at 2 and 24 h. Similar ROI was designed over explanted tissues for ex vivo analysis.
Data were analyzed with GraphPad Prism statistical software 8.0 (GraphPad Software, La Jolla, CA, USA). In vitro results were obtained from at least three independent experiments and are expressed as means ± standard deviation and significance was determined using Student’s t test.
A p value < 0.05 was considered statistically significant. For in vivo results, normality was tested with a Shapiro–Wilk’s test, and parametric or non-parametric tests were chosen accordingly. Tumor volumes and bodyweights were studied over time by applying a mixed model analysis of variance for repeated measures (RM-ANOVA) with Geisser-Greenhouse correction, hence studying the effect of time, of the treatment group and of their interaction. Post hoc, Fisher’s LSD test for multiple comparisons was applied between groups. The FRI counts/energy, in vivo at 2 and 24 h and ex vivo, between treatment groups were tested with a Kruskal–Wallis test, and post hoc with a Dunn’s test for multiple comparisons.