SK-N-BE(2) (ATCC: no. CRL-2271) cells were cultured in MEM alpha (Lonza, Cat #12-349-015), supplemented with heat-inactivated FBS (Hyclone, Cat# SH3091003), sodium pyruvate (Lonza, Cat# BW13-115E), Antibiotic–Antimycotic (Gibco, Cat#15-240-062), beta-mercaptoethanol (Gibco, Cat#21-985-023), MEM NEAA (Gibco, Cat#11140-050), and 50 mg/mL Normocin (Invivogen, Cat# ant-nr-2) and maintained in a 37 °C humidified incubator with 5% CO2.
Animal studies were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh, and in accordance with the relevant guidelines and regulations (IACUC protocol number: 22101912 and ARRIVE guidelines). Five-to-six-week-old female athymic nude rats (nu/nu) (NIH-Foxn1rnu, strain code 316), purchased from Charles Rivers Laboratories, were maintained in a temperature-controlled animal facility at the UPMC Children’s Hospital of Pittsburgh with a 12-h light/dark cycle. Animals were kept in the facility for at least two days prior to performing any procedures. Animals were monitored every other day and weighed at least weekly. Humane endpoint was defined as a loss of more than 20% of the original body weight; signs of significant distress from tumor burden such as decreased activity, inability to walk normally, substantially distended abdomen, or body condition score less than 2; or tumor size greater than 4 cm in its largest dimension or 5000 mm3 in volume as seen on MRI (per direction by the University of Pittsburgh veterinary group and American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020))19. Animals were euthanized in a carbon dioxide chamber followed by confirmatory cervical dislocation within 12 h of reaching humane endpoint (per approved IACUC protocol in accordance with Human Euthanasia practices).
Tumors were grafted as previously described in mice, but in 5–6-week-old female RNU homozygous rats (Charles Rivers Laboratories)20. SK-N-BE(2) neuroblastoma cells at 70–90% confluence were trypsinized, resuspended in PBS, and counted (10µL mixed with Trypan blue and counted with automated hemocytometer). 1.9 × 106 SK-N-BE(2) neuroblastoma cells in 30 µL of PBS were mixed with 30 µL of Matrigel basement membrane matrix (Corning Cat#354234; Fig. 1A), and pipetted into a 1 cc syringe with a 23-gauge needle for each rat. Under isoflurane anesthesia (using SomnoFlow machine), each rat was placed in right lateral decubitus position with a small bump (i.e. folded gauze) under the upper abdomen, hair was trimmed if necessary, a transverse left flank incision was made through the skin and abdominal wall muscle, and the left kidney and adrenal gland were exteriorized with gentle external pressure and a sterile cotton tip applicator (Fig. 1A). Matrigel-suspended cells were then implanted into the left adrenal gland and surrounding fat pad before the needle was slowly removed (Fig. 1A). The kidney and adrenal gland were returned to the abdominal cavity, the body wall was closed with 3–0 or 4–0 Polysorb suture, and the skin was closed with surgical wound clips. The animals were given meloxicam for analgesia, recovered in a clean cage on a heating pad, and were monitored until awake and active. Wound clips were removed 10–14 days later. Rats that did not have appreciable tumors within 6.5 weeks were removed from further analysis (1 out of 6 rats in the survival group and 3 out of 12 rats in the surgery group).
Survival and MRI
Following tumor injection, 5–6-week-old rats (n = 6, no surgery cohort) were monitored for growth, symptoms, and tumor development. MRIs were done twice weekly, beginning 3.5 weeks after tumor implantation. Due to the intraperitoneal location of the tumors, size could not be measured externally. Rats were euthanized when the humane endpoint was reached (see “Animal studies” section). One rat did not have an appreciable tumor on MRI 6.5 weeks after implantation and was excluded from further analysis. For the surgery cohort (n = 9), MRIs were performed 5 weeks after tumor injection to confirm adequate growth prior to surgery, then again when rats exhibited symptoms of recurrence.
To obtain each MRI, rats were anesthetized via a nose cone with 1–2% isoflurane and 3L/m O2, positioned on an animal bed, and placed in the scanner. Respiration rate was monitored, and body temperature was maintained using a warm air heating system, (SA Instruments, New York, NY, USA). MRI was performed using a 7 T/30-cm AVIII spectrometer (Bruker Biospin, Billerica, MA) equipped with a 12 cm gradient set and using an 86 mm quadrature RF volume coil and Paravision 6.0.1. Anatomical scans on the abdomen were performed using a self-navigating Intragate sequence with retrospective gating on the respiration with the following parameters: repetition time (TR)/echo time (TE) = 200/2.6, field of view (FOV) = 70 × 70 mm, acquisition matrix = 256 × 256, 19 coronal slices with a slice thickness of 1.5 mm. Each scan was performed over a period of 25 min.
Tumor resection (surgery)
Neuroblastoma tumors were implanted in 5–6-week-old rats (n = 12), as described above. MRI was done 5 weeks after injection and resection was attempted in rats with tumors greater than 0.7 cm in largest dimension (n = 9). Tumor volume was calculated from MRI images using DSI studios DICOM analysis. Three rats did not have appreciable tumors at this time and were excluded from further analysis.
Under 2–3% isoflurane anesthesia and 3L/m O2 (administered by a research assistant), the rat was laid supine while respirations were monitored, and body temperature was maintained using a heating pad. A lateral transverse abdominal incision was made (Fig. 3A) and sharp dissection combined with a fine-tip cautery tool (BVI Accu Temp Cat# D901) was used to open the abdominal wall and meticulously separate the tumor from the surrounding healthy tissue. Surgical loupes were typically used by the surgeon for additional magnification. Additional tumor nodules were resected when observed. The peritoneum was closed with 3–0 or 4–0 suture and skin was closed with skin clips, which were removed 10–14 days after surgery. Topical lidocaine spray was sprayed over the incision and 1 mg/kg of meloxicam was given via intraperitoneal injection. Rats did not show signs of pain following surgery nor in the following days. Rats were followed for tumor recurrence and were euthanized when the humane endpoint was reached.
Tumor samples were fixed in 10% neutral buffered formalin, paraffin-embedded, cut, and stained with hematoxylin and eosin21. Immunohistochemical staining for PHOX2B was performed on the Ventana Benchmark Ultra automated staining platform. Slides were pretreated with ultraCC1 (proprietary, Roche Tissue Diagnostics, Indianapolis, IN) and stained using a recombinant antibody to PHOX2B (EPR14423) (Abcam, Cambridge, MA, ab183741). OptiView DAB detection kit (proprietary, indirect, biotin free system, Roche Tissue Diagnostics, Indianapolis, IN) was used for primary antibody detection. All slides were counterstained with hematoxylin and routinely dehydrated, cleared, and cover-slipped in resinous mounting medium.
All statistical analyses were performed with GraphPad Prism (version 10.0.2 for Windows, GraphPad Software, Boston, MA). Tumor sizes were compared using an unpaired t test. Kaplan–Meier analysis with log-rank Mantel-Cox test was used to compare survival of untreated neuroblastoma model rats and neuroblastoma model rats that underwent surgical resection. Simple linear regression was used to assess correlation between tumor volumes on MRI and by caliper measurement, as well as resected and recurrent tumor sizes.
Ethics approval and consent to participate
Animal studies were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh (IACUC protocol number: 22101912).