TITLE: Neural recordings of spontaneously metastasizing melanomas and melanomas with low metastatic potential
AUTHORS: Jay Shiralkar, Tiana Anthony, Grant A McCallum, Dominique Durand

[DESCRIPTION]
Multiple studies report that melanomas are innervated tumors with sensory and sympathetic fibers where these neural fibers play crucial functional roles in tumor growth and metastasis with branch specificity. Yet there is no study which reports the direct neural recording and its pattern during in-vivo progression of the cancer. We performed daily neural recordings from male and female mice bearing orthotopic metastasizing-melanomas and melanomas with low metastatic poential, derived from B16-F10 and B16-F1 cancer cells, respectively. Further, to explore the origins of neural activity, 6-Hydroxidopamine mediated chemical sympathectomy was performed followed by daily microneurographic recordings. We also performed the daily bioluminescent imaging to track in vivo growth of primary tumors and distant metastasis to the cranial area. The protocols contained herein describe the methods, materials and equipment used to accomplish this research.

[STEPS]
SECTION: B16-F10-Luc2 &amp; B16-F1-Luc2 Cell Culturing Protocol
1. Store the vials containing cells in liquid nitrogen vapor until they are ready for use.
SECTION: B16-F10-Luc2 &amp; B16-F1-Luc2 Cell Culturing Protocol
2. Thaw in a 37°C water bath, then decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on will be carried out under strict aseptic conditions. 
SECTION: B16-F10-Luc2 &amp; B16-F1-Luc2 Cell Culturing Protocol
3. Add vial contents to a complete culture medium, and then centrifuge at approximately 125 x g for 5 to 7 minutes.
SECTION: B16-F10-Luc2 &amp; B16-F1-Luc2 Cell Culturing Protocol
4. Add the cell pellet to the recommended complete medium. Medium: DMEM, 10% FBS, 1% Penicillin-Streptomycin 
SECTION: B16-F10-Luc2 &amp; B16-F1-Luc2 Cell Culturing Protocol
5. Incubate the culture at 37°C in a suitable incubator at proper CO2 exposure: 5% CO2 
SECTION: B16-F10-Luc2 &amp; B16-F1-Luc2 Cell Culturing Protocol
6. Renew medium every 2- 3 days: remove and discard old medium. 
SECTION: B16-F10-Luc2 &amp; B16-F1-Luc2 Cell Culturing Protocol
7. Rinse cell layer with 1X PBS 
SECTION: B16-F10-Luc2 &amp; B16-F1-Luc2 Cell Culturing Protocol
8. Add 2.0 to 3.0 mL of Trypsin solution to 75cm3 flask and observe cells under an inverted microscope until the cell layer is dispersed (usually within 15 minutes). Corning‱ T-75 flasks (catalog #430641) are recommended for subculturing this product.
SECTION: B16-F10-Luc2 &amp; B16-F1-Luc2 Cell Culturing Protocol
9. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting
SECTION: B16-F10-Luc2 &amp; B16-F1-Luc2 Cell Culturing Protocol
10. Add appropriate aliquots of the cell suspension to new culture vessels.  Cultures can be established between 2 x 104 and 4 x 104 viable cells/cm2
SECTION: B16-F10-Luc2 &amp; B16-F1-Luc2 Cell Culturing Protocol
11. Incubate cultures at 37°C and 5% CO2 
SECTION: B16-F10-Luc2 &amp; B16-F1-Luc2 Cell Culturing Protocol
12. Passage cells at least twice after thawing and before inoculation, but no more than 30 times total per cell
line. 
SECTION: B16-F10-Luc2 &amp; B16-F1-Luc2 Cell Culturing Protocol
13. Under isoflurane anesthesia (2%, 1L/min), each mouse underwent a subcutaneous injection with 5 x 105 cells to the flank area. 
SECTION: Chemical sympathectomy
14. Chemical sympathectomy solution was prepared by dissolving 6-hydroxydopamine (6-OHDA, 100 mg/kg body weight) in sterile saline along with 0.01% of ascorbate as a stabilizer.   2 mL of buffer per 5 mg vial provides a solution containing 10 mM 6-hydroxydopamine and 0.01% (w/v) ascorbic acid.
SECTION: Chemical sympathectomy
15. On the fourth (Day = -4) and second day (Day = -2) before cancer cell inoculation (Day = 0), the drug was injected intraperitoneally (IP) using an insulin syringe. 
SECTION: Chemical sympathectomy
16. After these two initial injections, the drug was administered IP every five days to maintain the sustained effect of denervation. (i.e. Day = +5, +10, +15, etc)
SECTION: Microneurography and neural recordings
17. Neural recordings were performed for thirty minutes for each day under 2% isoflurane in 1 L/min oxygen, for two weeks starting from day 6 post inoculation of cancer cells. 
SECTION: Microneurography and neural recordings
18. Microneurography needles were inserted into the primary tumor mass and a ground/reference needle was inserted on the contralateral side.
SECTION: Microneurography and neural recordings
19. By using a micromanipulator, the needles were inserted at the same depth (5 mm) and same distance apart (4 mm), daily, in order to record from the same location. The two recording needles were fixed to a holder and were maintained at a 4 mm separation distance. The holder was mounted on a 1µm, x-y-z resolution micromanipulator system, so that the daily insertion depth could be repeated with certainty. 
SECTION: Microneurography and neural recordings
20. The neural data were sampled at 20 kHz and filtered from 500 Hz to 1200 Hz using a 7th order, zero-phase, digital bandpass filter.
ADInstruments amplifier (FE285) was used for all the recordings. Recorded neural data were displayed and stored in Lab Chart files. 
SECTION: Microneurography and neural recordings
21. Lab Chart files were exported into MATLAB (RRID:SCR_001622) compatible files.
SECTION: Microneurography and neural recordings
22. The results were quantified in terms of number of spikes per file using UltraMegaSort2000 (UMS2000) spike sorting program (2) and neural spikes were plotted for ten minutes. 
SECTION: Microneurography and neural recordings
23. The averaged spike count per 10 minutes, was plotted as a function of days post inoculation of cancer cells.
SECTION: Bioluminescence imaging
24. Images were acquired using Perkin-Elmer’s In-Vivo Imaging System (IVIS) Spectrum at 60s exposure. 
SECTION: Bioluminescence imaging
25. Under isoflurane anesthesia (2% at 1L/min), 200 µL of D-luciferin solution (12.5 mg/ml of D-luciferin in sterile phosphate buffer solution) was injected IP using an insulin syringe ten minutes before acquiring the images. 
SECTION: Bioluminescence imaging
26. Total flux was quantified from the primary tumor region in order to quantify the in-vivo growth, as well as from the cranial region to quantify the growth of secondary metastatic foci. 
SECTION: Bioluminescence imaging
27. Same sized regions of interest (ROIs) were used for the images with the same Field of View (FOVs) for a particular day of imaging.   
SECTION: Histology
28. At day 10 post-inoculation, under isoflurane anesthesia (2%, 1L/min) the mice were perfused via intracardiac perfusion first using 20 mL of 1X Phosphate Buffered Saline (PBS) solution followed by 20 mL of 4% Paraformaldehyde (PFA) solution. 
SECTION: Histology
29. The excised tumor tissues were then transferred to paraformaldehyde and refrigerated at 4°C for 24 to 48 hours. 
SECTION: Histology
30. After fixing in paraformaldehyde, the samples were immersed in a 15% sucrose solution with 1X PBS for 24 hours at 4°C. 
SECTION: Histology
31. Then, they were moved to a 30% sucrose 1X PBS solution and kept at 4°C until processing
SECTION: Histology
32. The samples were formalin-fixed and paraffin-embedded, and 5μm thick slices were subjected to immunohistochemistry chromogenic detection using neurofilament primary antibody (NF, RRID: Thermo Fisher 2F11; TH, RRID: Sigma-Aldrich AB152) to detect the presence of nerve fibers and autonomic nerves, respectively. 

*Tissues were fixed in 10% NBF , were embedded in paraffin, sectioned and mounted on slides , and then IHC stained.

 
 
 

SECTION: Histology
33. To obtain a comprehensive pathological understanding of the tumor microenvironment, slices were obtained from various tumor areas, ranging from about 100 μm. 
SECTION: Histology
34. The neurofilament immunohistochemical (IHC) staining process was conducted as follows:
SECTION: Histology
34.1. Neurofilament Protocol:
Involved in the maintenance of neuronal caliber, neurofilaments are the intermediate filament proteins found specifically in neurons, and are composed predominantly of three major proteins called NF-L, NF-M and NF-H. Like most other intermediate filament proteins (IFPs), the expression of the different neuronal IFPs is both tissue-specific and developmentally regulated. NF-L is the light or low molecular weight microfilament subunit and runs on SDS-PAGE gels at approximately 70 kDa. Neurofilament are the 10nm or intermediate filament proteins found specifically in neurons, and are composed predominantly of three major proteins called NF-L, NF-M and NF-H. NF-H is the heavy or high molecular weight microfilament subunit and runs on SDS-PAGE gels in the range 180-220 kDa, with some variation in different species.
SECTION: Histology
35. The tyrosine hydroxylase (TH) immunohistochemical (IHC) staining process was conducted as follows:
SECTION: Histology
34.2. Bake slides at 60ºC for 75 min. (let cool for 20 min. after time elapses)
SECTION: Histology
34.3. Deparaffinize slides in xylene, 2X  for 7 min. each time
SECTION: Histology
34.4. Rehydrate slides in graded ethanols: 
          a.  100% ethanol, 2X for 2 min. each time 
          b.  95% ethanol, 2X for 2 min. each time 
          c.  70% ethanol for 2 min.
SECTION: Histology
34.5. Rinse slides in dH2O. for 2 min. or use squirt bottle
SECTION: Histology
34.6. [10X] Antigen Unmasking Solution/ Citric Acid Based- pH 6.0/ Vector Labs/ Cat#: H-3300 

Antigen retrieval: 
          a.   Place slides in 250 mL [1X] Antigen Unmasking Buffer 
          b.   Incubate in pressure cooker at 120 ºC for 30 seconds 
          c.   Remove slides and let cool on bench top for 20 min.
SECTION: Histology
34.7. Rinse slides in dH2O for 2 min. 
SECTION: Histology
34.8. Peroxidazed 1: (Ready-to-Use) / BioCare Medical / Cat#: PX968G

Block endogenous peroxidase activity: 
          a.	Place Slides in Peroxidazed 1 for 8 min.
SECTION: Histology
34.9. Rinse slides in dH2O for 2 min. 
SECTION: Histology
34.10. Rodent Block M: (Ready-to-Use) / BioCare Medical/ Cat#: RBM961H / 

Block endogenous mouse IgG: 
            a.	Place slides in Rodent Block M for 30 min.
SECTION: Histology
34.11. Rinse slides in TBST 
SECTION: Histology
34.12. Mouse anti-Neurofilament (NEFL):Mouse Monoclonal Antibody IgG1 /Invitrogen/ Cat#:MA1-06803/ Clone: 2F11/ 

Apply primary antibody 
            a.   [1:200] =  X µL Neurofilament in  X µL Diluent 
            b.   Incubate slides at room temp. for one hour.
SECTION: Histology
34.13. Rinse slides in TBST 
SECTION: Histology
34.14. Mouse-on-Mouse HRP-Polymer: (Ready-to-Use) / Biocare Medical /Cat#: MM620G

Apply Mouse on Mouse HRP-Polymer for 30 min.
SECTION: Histology
34.15. Rinse slides in TBST
SECTION: Histology
34.16. Betazoid DAB: BioCare Medical / Cat#: BDB2004H 

Apply chromogen: 
           a.  1 drop DAB in 1 mL dH2O 
           b.  Incubate in Betazoid DAB for 5 min.
SECTION: Histology
34.17. Rinse slides in dH2O
SECTION: Histology
34.18. CAT Hematoxylin: (Ready-to-Use) / BioCare Medical / Cat#: CATHE-L /  Lot no. 12513B

Counterstain with CAT Hematoxylin for1 min.
SECTION: Histology
34.19. Rinse in running tap water for 30 seconds
SECTION: Histology
34.20. Rinse in dH2O 3X for 1 min. each time
SECTION: Histology
34.21. Blue the nuclei with ammonia water by dipping ten times
SECTION: Histology
34.22. Rinse in running tap water for 30 seconds
SECTION: Histology
34.23. Coverslip with resinous medium
SECTION: Histology
34.24. Number of events per square millimeter of tissue slices were calculated in order to quantify and compare the staining metrics. 
SECTION: Histology
35.1. Tyrosine Hydroxylase plays an important role in the physiology of adrenergic neurons. It is the first and rate-limiting enzyme involved in the biosynthesis of the catecholamines Dopamine and Norepinephrine from tyrosine. TH is, therefore, a useful marker for dopaminergic and noradrenergic neurons. The enzymatic activity of TH requires ferrous ions as cofactors and is believed to be regulated by phosphorylation. At least four isoforms of human TH have been identified which result from alternative splicing.
SECTION: Histology
35.2. Bake slides at 60ºC for 75 min.
SECTION: Histology
35.3. Deparaffinize slides in xylene, 2X for 7 min. each time 
SECTION: Histology
35.4. Rehydrate slides in graded ethanols:
                                    a.  100% ethanol, 2X for 2 min. each time
                                    b.  95% ethanol, 2X for 2 min. each time
                                    c.  70% ethanol for 2 min.
SECTION: Histology
35.5. Rinse slides in dH2O
SECTION: Histology
35.6. X Rodent Decloaker (pH 6.6): Biocare Medical / Cat#:RD913M / Lot no. 101608 / (To be diluted to 1 X)

Antigen retrieval:
                                    a.   Place slides in 250 mL 1X Rodent Buffer                  
                                    b.   Incubate in Steamer at ~98°C for 20 minutes
                                    c.   Remove slides and let cool on bench top for 20 min.
SECTION: Histology
35.7. Rinse slides in dH2O 
SECTION: Histology
35.8. Peroxidazed 1: (Ready-to-Use / BioCare Medical / Cat#: PX968M / Lot no. no070109-1
 
Block endogenous peroxidase activity:
                                    a.  Place Slides in Peroxidazed 1 for 8 min.
SECTION: Histology
35.9. Rinse slides in dH2O  
SECTION: Histology
35.10. Rodent Block M: (Ready-to-Use)/ BioCare Medical /Cat#: RBM961 /  Lot no. 091009-1 (MOUSE TISSUES ONLY

Block endogenous mouse IgG and non-specific background staining:
                                    a.   Place select slides in Rodent Block M for 20 min.
SECTION: Histology
35.11. Rinse slides in TBST 
SECTION: Histology
35.12. Rabbit Polyclonal Tyrosine Hydroxylase Antibody: Millipore/ Cat#: AB152/ Lot no. JC1682878

Apply primary antibodies
                                 a.      [1:500] =  X µL of TH stock in X µL Diluent
                                 b.     Incubate slides overnight at 4° C.
SECTION: Histology
35.13. Rinse slides in TBST 
SECTION: Histology
35.14. Rabbit-on-Rodent HRP Polymer: (Ready-to-Use)/ BioCare Medical/ Cat#:RMR622H/ Lot no. 010214

Apply Rabbit Polymer HRP for 30 min.
SECTION: Histology
35.15. Rinse slides in TBST
SECTION: Histology
35.16. Betazoid DAB: BioCare Medical / Cat#: BDB2004L / Lot no. 092509
 
Apply chromogen:
                                    a.   Incubate in Betazoid DAB for 5 min.
                                    b.   1 drop DAB in 1.0 mL dH2O
SECTION: Histology
35.17. Rinse slides in dH2O 
SECTION: Histology
35.18. CAT Hematoxylin: (Ready-to-Use) / BioCare Medical / Cat#: CATHE-L /  Lot no. 12513B
 
Counterstain with CAT Hematoxylin for 30 secs.
SECTION: Histology
35.19. Rinse in running tap water for 30 seconds
SECTION: Histology
35.20. Rinse in dH2O 3X for 1 min. each time
SECTION: Histology
35.21. Blue the nuclei with TBST wash buffer   
SECTION: Histology
35.22. Rinse in running tap water for 30 seconds 
SECTION: Histology
35.23. Air dry overnight or dehydrate to Xylene
SECTION: Histology
35.24. Coverslip with resinous medium
SECTION: Histology
35.25. Number of events per square millimeter of tissue slices were calculated in order to quantify and compare the staining metrics.