Neural recordings of spontaneously metastasizing melanomas and melanomas with low metastatic potential

Store the vials containing cells in liquid nitrogen vapor until they are ready for use.

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.

Add vial contents to a complete culture medium, and then centrifuge at approximately 125 x  g  for 5 to 7 minutes.

Add the cell pellet to the recommended complete medium. Medium: DMEM, 10% FBS, 1% Penicillin-Streptomycin

Incubate the culture at 37°C in a suitable incubator at proper CO₂ exposure: 5% CO₂

Renew medium every 2- 3 days: remove and discard old medium.

Rinse cell layer with 1X PBS

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.

Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting

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

Incubate cultures at 37°C and 5% CO₂

Passage cells at least twice after thawing and before inoculation, but no more than 30 times total per cell

line.

Under isoflurane anesthesia (2%, 1L/min), each mouse underwent a subcutaneous injection with 5 x 10⁵ cells to the flank area.

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.

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.

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)

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.

Microneurography needles were inserted into the primary tumor mass and a ground/reference needle was inserted on the contralateral side.

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.

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.

Lab Chart files were exported into MATLAB (RRID:SCR_001622) compatible files.

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.

The averaged spike count per 10 minutes, was plotted as a function of days post inoculation of cancer cells.

Images were acquired using Perkin-Elmer’s In-Vivo Imaging System (IVIS) Spectrum at 60s exposure.

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.

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.

Same sized regions of interest (ROIs) were used for the images with the same Field of View (FOVs) for a particular day of imaging.  

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.

The excised tumor tissues were then transferred to paraformaldehyde and refrigerated at 4°C for 24 to 48 hours.

After fixing in paraformaldehyde, the samples were immersed in a 15% sucrose solution with 1X PBS for 24 hours at 4°C.

Then, they were moved to a 30% sucrose 1X PBS solution and kept at 4°C until processing

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.

A	B	C	D	E	F	G	H	I
Name	RRID	Source or reference	Catalog number	Antibody type	Target	Raised in	Clonality	Dilution used
NEFL Monoclonal Antibody (2F11)	RRID:AB_560286	Thermo Fisher Scientific	MA1-06803	primary	NEFL	mouse	monoclonal	1:200
Anti-Tyrosine Hydroxylase Antibody	RRID:AB_390204	Millipore	AB152	primary	Tyrosine Hydroxylase	rabbit	polyclonal	1:500

To obtain a comprehensive pathological understanding of the tumor microenvironment, slices were obtained from various tumor areas, ranging from about 100 μm.

The neurofilament immunohistochemical (IHC) staining process was conducted as follows:

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.

Rodent Block M: (Ready-to-Use) / BioCare Medical/ Cat#: RBM961H /

Block endogenous mouse IgG:

1.	Place slides in Rodent Block M for 30 min.

Rinse slides in TBST

Mouse anti-Neurofilament (NEFL):Mouse Monoclonal Antibody IgG1 /Invitrogen/ Cat#:MA1-06803/ Clone: 2F11/ ₁ /Invitrogen/ Cat#:MA1-06803/ Clone: 2F11/

Apply primary antibody

1.   [1:200] =  X µL Neurofilament in  X µL Diluent 

2.   Incubate slides at room temp. for one hour.

Rinse slides in TBST

Mouse-on-Mouse HRP-Polymer: (Ready-to-Use) / Biocare Medical /Cat#: MM620G

Apply Mouse on Mouse HRP-Polymer for 30 min.

Rinse slides in TBST

Betazoid DAB: BioCare Medical / Cat#: BDB2004H

Apply chromogen:

1.  1 drop DAB in 1 mL dH<sub>2</sub>O 



2.  Incubate in Betazoid DAB for 5 min.

Rinse slides in dH₂O

CAT Hematoxylin: (Ready-to-Use) / BioCare Medical / Cat#: CATHE-L / Lot no. 12513B

Counterstain with CAT Hematoxylin for1 min.

Rinse in running tap water for 30 seconds

Bake slides at 60ºC for 75 min. (let cool for 20 min. after time elapses)

Rinse in dH2O 3X for 1 min. each time

Blue the nuclei with ammonia water by dipping ten times

Rinse in running tap water for 30 seconds

Coverslip with resinous medium

Number of events per square millimeter of tissue slices were calculated in order to quantify and compare the staining metrics.

Deparaffinize slides in xylene, 2X for 7 min. each time

Rehydrate slides in graded ethanols:

1.  100% ethanol, 2X for 2 min. each time 

2.  95% ethanol, 2X for 2 min. each time 

3.  70% ethanol for 2 min.

Rinse slides in dH₂O. for 2 min. or use squirt bottle

[10X] Antigen Unmasking Solution/ Citric Acid Based- pH 6.0/ Vector Labs/ Cat#: H-3300

Antigen retrieval:

1.   Place slides in 250 mL [1X] Antigen Unmasking Buffer 

2.   Incubate in pressure cooker at 120 ºC for 30 seconds 

3.   Remove slides and let cool on bench top for 20 min.

Rinse slides in dH₂O for 2 min.

Peroxidazed 1: (Ready-to-Use) / BioCare Medical / Cat#: PX968G

Block endogenous peroxidase activity:

1.	Place Slides in Peroxidazed 1 for 8 min.

Rinse slides in dH2O for 2 min.

The tyrosine hydroxylase (TH) immunohistochemical (IHC) staining process was conducted as follows:

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.

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.

Rinse slides in TBST

Rabbit Polyclonal Tyrosine Hydroxylase Antibody: Millipore/ Cat#: AB152/ Lot no. JC1682878

Apply primary antibodies

1.      [1:500] =  X µL of TH stock in X µL Diluent

2.     Incubate slides overnight at 4° C.

Rinse slides in TBST

Rabbit-on-Rodent HRP Polymer: (Ready-to-Use)/ BioCare Medical/ Cat#:RMR622H/ Lot no. 010214

Apply Rabbit Polymer HRP for 30 min.

Rinse slides in TBST

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

Rinse slides in dH₂O

CAT Hematoxylin: (Ready-to-Use) / BioCare Medical / Cat#: CATHE-L / Lot no. 12513B

Counterstain with CAT Hematoxylin for 30 secs.

Rinse in running tap water for 30 seconds

Bake slides at 60ºC for 75 min.

Rinse in dH₂O 3X for 1 min. each time

Blue the nuclei with TBST wash buffer  

Rinse in running tap water for 30 seconds

Air dry overnight or dehydrate to Xylene

Coverslip with resinous medium

Number of events per square millimeter of tissue slices were calculated in order to quantify and compare the staining metrics.

Deparaffinize slides in xylene, 2X for 7 min. each time

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.

Rinse slides in dH₂O

X Rodent Decloaker (pH 6.6): Biocare Medical / Cat#:RD913M / Lot no. 101608 / (To be diluted to 1 X)

Antigen retrieval:

1.   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.

Rinse slides in dH₂O

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.

Rinse slides in dH₂O