Jul 23, 2024
Neural recordings of spontaneously metastasizing melanomas and melanomas with low metastatic potential
- Jay Shiralkar1,
- Tiana Anthony1,
- Grant A McCallum1,
- Dominique Durand1
- 1Case Western Reserve University
Protocol Citation: Jay Shiralkar, Tiana Anthony, Grant A McCallum, Dominique Durand 2024. Neural recordings of spontaneously metastasizing melanomas and melanomas with low metastatic potential. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbzn7ngpk/v1
Manuscript citation:
Shiralkar J, Anthony T, McCallum GA, Durand DM (2024) Neural recordings can differentiate between spontaneously metastasizing melanomas and melanomas with low metastatic potential. PLoS ONE 19(2): e0297281. https://doi.org/10.1371/journal.pone.0297281
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 18, 2024
Last Modified: July 23, 2024
Protocol Integer ID: 102033
Keywords: Melanoma, tumors, sympathetic nervous system, chronic recording, metastasis, autonomic nervous system, microneurography, murine, mouse, neuroelectric activity
Funders Acknowledgement:
CDMRP - BCRP
Grant ID: W81XWH-18-1-0581
Abstract
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.
Materials
MATERIALS
| A | B | C | D | E | |
| Protocol | Description | Manufacturer | Part Number | RRID | |
| A. Mouse Models | |||||
| C57BL/6J mice | Jackson Laboratories | RRID:IMSR_JAX:000664 | |||
| B16-F10 | ATCC | CRL-6475-LUC2 | RRID:CVCL_A4CJ | ||
| B16-F1 | ATCC | CRL-6323-LUC2 | RRID:CVCL_A4CK | ||
| Dulbecco's Modified Eagle's Medium (DMEM) | ATCC | 30-2002 | |||
| 10% Fetal Bovine Serum (FBS) | ATCC | 30-2020 | |||
| 1% Penicillin-Streptomycin Solution | ATCC | 30- 2300 | |||
| 1X Phosphate Buffered Saline (PBS) (diluted) | Teknova | P3195 | |||
| 4% Paraformaldehyde (PFA) | Thermo Fisher | J19943.K2 | |||
| B. Chemical Sympathectomy | |||||
| 6-hydroxydopamine hyrobromide (6-OHDA) | Sigma-Aldrich | H116 | |||
| C. Neural Recordings | |||||
| Isoflurane | Covetrus | 29405 | |||
| D. Bioluminescence Imaging | |||||
| D-luciferin salt | Gold Biotechnology | LUCK-1G | |||
| 1X Phosphate Buffered Saline (PBS) (diluted) | Teknova | P3195 | |||
| E. Histology | |||||
| 1X Phosphate Buffered Saline (PBS) (diluted) | Teknova | P3195 | |||
| 4% Paraformaldehyde (PFA) | Thermo Fisher | J19943.K2 | |||
| Sucrose | Sigma-Aldrich | S0389 | |||
| Neurofilament antibody (NF) | Thermo Fisher | 2F11 | RRID:AB_560286 | ||
| Tyrosine Hydroxylase antibody (TH) | Sigma-Aldrich | AB152 | RRID:AB_390204 | ||
| Antigen Unmasking Solution, Citrate-Based - pH 6.0 | Vector Labs | H-3300-250 | |||
| Peroxidazed 1 (Ready-to-Use) | BioCare Medical | PX968G | |||
| Rodent Block M | BioCare Medical | RBM961H | |||
| Mouse-on-Mouse HRP-Polymer | BioCare Medical | MM620G | |||
| Betazoid DAB | BioCare Medical | BDB2004H | |||
| CAT Hematoxylin (Ready-to-Use) | BioCare Medical | CATHE-L; Lot no. 12513B | |||
| Rodent Decloaker, 10X, pH 6.6 | BioCare Medical | RD913M / Lot no. 101608 | |||
| Peroxidazed 1, Ready-to-Use | BioCare Medical | PX968M / Lot no. no070109-1 | |||
| Rodent Block M: (Ready-to-Use) | BioCare Medical | RMB961/ Lot no. 091009-1 (Mouse tissues only) | |||
| Rabbit-on-Rodent HRP Polymer: (Ready-to-Use) | BioCare Medical | RMR622H/ Lot no. 010214 | |||
| Betazoid DAB | BioCare Medical | BDB2004L / Lot no. 092509 |
EQUIPMENT
| A | B | C | D | |
| Protocol | Description | Manufacturer | Part Number | |
| A. Mouse Models | ||||
| Anesthesia Machine | Parkland Scientific | SN. 1172 | ||
| B. Chemical Sympathectomy | ||||
| Insulin syringes | Carepoint Vet | 12-7903 | ||
| C. Neural Recordings | ||||
| Micromanipulator | WPI | M3301R | ||
| Microneurography needle (reference) | FHC | 30084 | ||
| Microneurography needle (active) | FHC | 30080 | ||
| Neural Amplifier (Neuro Amp EX) | AD Instruments | FE285 | ||
| Neuro Amp EX Headstage | AD Instruments | MLT185 | ||
| Signal Acquisition System | AD Instruments | PowerLab 8/35 | ||
| Signal Storage and Visualization Software | AD Instruments | LabChart v8.1.13 | ||
| Spike Sorting Software | Citation (2) | UltraMegaSort 2000 | ||
| D. Bioluminescence Imaging | ||||
| BLI imaging system | Perkin-Elmer | In-Vivo Imaging System (IVIS) | ||
| Insulin syringes | Carepoint Vet | 12-7903 | ||
| E. Histology | ||||
| 20mL syringes | Nipro | JD+20L |
B16-F10-Luc2 & B16-F1-Luc2 Cell Culturing Protocol
B16-F10-Luc2 & B16-F1-Luc2 Cell Culturing Protocol
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
Chemical sympathectomy
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)
Microneurography and neural recordings
Microneurography and neural recordings
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.
Bioluminescence imaging
Bioluminescence imaging
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.
Histology
Histology
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.
Bake slides at 60ºC for 75 min. (let cool for 20 min. after time elapses)
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. for 2 min. or use squirt bottle
[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.
Rinse slides in dH2O for 2 min.
Peroxidazed 1: (Ready-to-Use) / BioCare Medical / Cat#: PX968G
Block endogenous peroxidase activity:
a. Place Slides in Peroxidazed 1 for 8 min.
Rinse slides in dH2O for 2 min.
Rodent Block M: (Ready-to-Use) / BioCare Medical/ Cat#: RBM961H /
Block endogenous mouse IgG:
a. 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/
Apply primary antibody
a. [1:200] = X µL Neurofilament in X µL Diluent
b. 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:
a. 1 drop DAB in 1 mL dH2O
b. 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
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.
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.
Bake slides at 60ºC for 75 min.
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:
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.
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
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
a. [1:500] = X µL of TH stock in X µL Diluent
b. 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
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.
Protocol references
1. Fazakas C, Wilhelm I, Nagyoszi P, Farkas AE, Haskó J, Molnár J, et al. Transmigration of melanoma cells through the blood-brain barrier: role of endothelial tight junctions and melanoma-released serine proteases. PloS One. 2011;6(6):e20758.
2. Hill DN, Mehta SB, Kleinfeld D. Quality Metrics to Accompany Spike Sorting of Extracellular Signals. J Neurosci. 2011 Jun 15;31(24):8699–705.