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Articolo metodologico

Imaging e quantificazione delle cellule immunitarie nel microambiente tumorale utilizzando un modello di neuroblastoma in zebrafish

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DOI:

10.3791/72028

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18 settembre 2026

 ,  ,  ,  ,  , 
1Departments of Pharmacology and Medicine, Section of Hematology and Medical Oncology, The Cancer Research Center, Boston University School of Medicine, 2Tulane University School of Medicine

* These authors contributed equally

In questo articolo

Sommario

Questo protocollo descrive metodi per generare zebrafish transgenici composti, per l'analisi di immagini di cellule immunitarie associate a tumori in zebrafish larvali e adulti, e per quantificare la frequenza delle cellule immunitarie all'interno del microambiente tumorale.

Abstract

Il reclutamento delle cellule immunitarie nell'ambiente tumorale (TME) rappresenta un componente fondamentale della regolazione immunitaria e della progressione tumorale. Lo studio delle relazioni spaziali e temporali tra cellule immunitarie e cellule tumorali in vivo può fornire informazioni dettagliate sulle interazioni all'interno del TME. Tuttavia, la visualizzazione e la quantificazione di queste interazioni in organismi integri rimangono complesse con approcci convenzionali di imaging e istologici. I pesci zebra costituiscono un modello prezioso per la ricerca oncologica grazie alla loro facilità di manipolazione genetica, allo sviluppo rapido e alla trasparenza ottica nelle fasi iniziali dello sviluppo, che permette la visualizzazione di cellule marcate con fluorescenza in vivo. Qui, presentiamo un protocollo per generare, identificare, visualizzare e quantificare cellule immunitarie e tumorali marcate in modo differenziale con marcatori fluorescenti in un modello di neuroblastoma nel pesce zebra. Il protocollo comprende l’allevamento e il screening di pesci zebra transgenici composti, l’imaging in vivo di larve, la preparazione e l’imaging di adulti portatori di tumore e la quantificazione delle cellule immunitarie circostanti e all’interno delle regioni tumorali. Utilizzando linee trasgeniche reporter, CD4+ cellule immunitarie e MYCN- le cellule di neuroblastoma possono essere visualizzate e analizzate a diverse fasi di sviluppo. Il flusso di lavoro consente la valutazione della localizzazione delle cellule immunitarie rispetto ai tumori sia nei pesci larvali che adulti e può essere utilizzato per confrontare l'abbondanza delle cellule immunitarie attraverso le diverse fasi della malattia. Questo protocollo fornisce un approccio riproducibile per l'acquisizione di immagini e la quantificazione delle cellule immunitarie all'interno dell'ambiente tumorale (TME) in vivo utilizzando il pesce zebra e potrebbe facilitare studi sulle interazioni tra sistema immunitario e tumore in altri modelli di cancro.

Introduzione

L'ambiente microambientale del tumore (TME) è un ecosistema composto da cellule tumorali, immunitarie, fibroblasti e vascolari1. Il TME svolge un ruolo essenziale nella progressione del cancro, nella risposta terapeutica e nell'immunità antitumorale. All'interno del TME, le cellule immunitarie sono regolatori chiave del destino del tumore, partecipando sia alle risposte antitumorali sia all'induzione di immunosoppressione2. Comprendere il reclutamento, la distribuzione e la funzione delle cellule immunitarie all'interno del TME è fondamentale per chiarire i meccanismi attraverso cui i tumori eludono il controllo immunitario. Tuttavia, gli approcci convenzionali allo studio del TME, come l'istologia endpoint, la citometria a flusso e il sequenziamento a singola cellula, forniscono soltanto istantanee statiche e spesso richiedono la distruzione della struttura del tessuto, con conseguente perdita di informazioni spaziali e interazioni tra cellule3,4,5,6,7. Inoltre, questi metodi presentano limitazioni nella capacità di visualizzare e quantificare le cellule immunitarie all'interno del TME, specialmente nelle fasi iniziali dello sviluppo del tumore.

Il pesce zebra (Danio rerio) è un modello potente per la ricerca sul cancro grazie al basso costo, allo sviluppo rapido, alla trasparenza relativa e all'elevata conservazione genetica8,9. Questi vantaggi forniscono un sistema sperimentale riproducibile e scalabile che permette ampie dimensioni campionarie e un efficace screening microscopico in un breve lasso di tempo10. In particolare, il pesce zebra condivide una significativa conservazione immunologica con l'uomo, inclusi componenti chiave sia del sistema immunitario innato che adattativo, rendendo i risultati pertinenti alle malattie umane11. Queste caratteristiche sono particolarmente utili per lo studio dei tumori pediatrici come il neuroblastoma8. Di conseguenza, sono state sviluppate numerose linee di pesce zebra con reporter immunitari e modelli di cancro11. Pertanto, il pesce zebra rappresenta un modello in vivo prezioso per lo studio delle interazioni tra cellule tumorali e immunitarie, offrendo spunti su come la regolazione immunitaria influenzi lo sviluppo del tumore, specialmente nelle fasi iniziali.

Studi precedenti che indagano il microambiente tumorale (TME) nel pesce zebra si sono basati ampiamente su modelli di xenotrapianto tumorale combinati con imaging a tempo finale per valutare la localizzazione delle cellule immunitarie all'interno dei tumori12,13,14,15,16,17. Sebbene questi metodi forniscano informazioni utili, sono limitati dall'utilizzo di immagini bidimensionali, che non riescono a rappresentare completamente l'organizzazione tridimensionale del TME e le relazioni spaziali tra cellule tumorali e immunitarie durante lo sviluppo14. Inoltre, i modelli di xenotrapianto tumorale implicano il trapianto artificiale di cellule tumorali umane e potrebbero non riprodurre fedelmente l'insorgenza naturale del tumore18. Questi modelli sono inoltre generalmente a breve termine e non riescono a catturare appieno il progresso tumorale o i cambiamenti nelle relazioni spaziali tra cellule tumorali e immunitarie durante lo sviluppo14,19. Inoltre, questo approccio fornisce una rappresentazione limitata dell'organizzazione spaziale, del contesto dello sviluppo e della rilevanza fisiologica del comportamento delle cellule immunitarie all'interno del TME. Recentemente, noi e altri ricercatori abbiamo utilizzato pesci zebra transgenici composti, nei quali le cellule tumorali e quelle immunitarie sono etichettate in modo differenziale con marcatori fluorescenti, per visualizzare le cellule immunitarie associate ai tumori20,21. Questi pesci transgenici composti permettono lo studio del TME durante tutto lo sviluppo dell'animale e del tumore, facilitando l'analisi delle interazioni tra sistema immunitario e tumore in vivo.

In questo studio, presentiamo un metodo che utilizza la microscopia confocale per analizzare le cellule immunitarie all'interno dell'ambiente tumorale (TME) di un modello di neuroblastoma nel pesce zebra. In questo approccio, abbiamo utilizzato linee di pesce zebra reporter che marcano le cellule immunitarie e una linea di pesce zebra geneticamente modificata che sovraesprime l'oncogene umano MYCN sotto il controllo del promotore della dopamina-β-idrossilasi (dβh), inducendo lo sviluppo di un neuroblastoma simile alla malattia umana20. In questo modello transgenico composto, le cellule tumorali e le cellule immunitarie sono marcate in modo differenziale con EGFP e mCherry, rispettivamente. Il promotore dβh è attivo nelle cellule della linea simpato-surrenale ed è stato utilizzato in diversi modelli di pesce zebra per lo studio del neuroblastoma22,23,24,25. Inoltre, abbiamo utilizzato un promotore di Cd4 che esprime la fluorescenza di mCherry per visualizzare le cellule CD4+15. L'acquisizione di immagini confocali con stack in Z è stata impiegata per visualizzare l'TME in tre dimensioni in diverse fasi dello sviluppo tumorale, consentendo di valutare le relazioni spaziali tra cellule tumorali e cellule immunitarie all'interno di tessuti intatti. Questo metodo permette di analizzare l'organizzazione delle cellule immunitarie rispetto alle cellule tumorali durante le diverse fasi dello sviluppo del tumore.

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Protocollo

All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Boston University Chobanian & Avedisian School of Medicine under protocol PROTO202100001. The overall workflow for generation of compound transgenic zebrafish, larval imaging, adult tissue preparation, and image analysis is summarized in Figure 1.

Zebrafish transgenic process diagram; fluorescence microscopy, embryo imaging, cryosection analysis.
Figure 1. Workflow for visualization and quantification of immune cells within the zebrafish tumor microenvironment (TME). Schematic overview of the experimental workflow. Immune-reporter zebrafish were crossed with tumor-prone zebrafish to generate compound transgenic offspring expressing fluorescent markers in both immune cells (red) and tumor cells (green). Embryos were screened by fluorescence microscopy to identify compound transgenic fish. Selected fish were either subjected to live confocal imaging during larval development (<21 days post-fertilization [dpf]) or processed by fixation, cryosectioning, and confocal imaging for fish older than 21 dpf. This workflow enables visualization and quantification of immune cells associated with the TME across multiple developmental stages. Part of the illustration was created using BioRender. Please click here to view a larger version of this figure.

1. Generation of Compound Transgenic Zebrafish Lines Through Breeding

  1. Select adult immune-reporter fish and tumor fish that are at least 3 months old for natural breeding.
    NOTE: For the representative results, Tg(cd4-1:mCherry) heterozygous fish were crossed with Tg(dβh:MYCN; dβh:EGFP) heterozygous fish to differentially label immune cells and tumor cells15,23.
  2. On the evening before breeding, fill a two-chamber mating tank two-thirds full with fish water.
  3. Place a grated top insert into the mating chamber. Use dividers to separate one male from one or two females.
  4. Leave the dividers in place overnight. Add artificial plants or other enrichment items to simulate natural breeding conditions.
    NOTE: A 1:2 male-to-female ratio increases the likelihood of successful spawning when breeding tumor male fish with immune-reporter fish. Do not breed two males with one female, as competition between males may occur.
  5. On the following morning, remove the dividers after the facility lights turn on.
  6. Tilt the grated top chamber slightly using the divider as a wedge to create a shallow water area. Allow the fish to spawn for at least 1 h.
  7. Collect eggs from the bottom of the mating chamber by pouring the water through a fine-mesh net.
  8. Transfer the eggs to a sterile Petri dish containing fish water at a density no greater than 1 egg per 50 mm2 (approximately 200 eggs per 100 mm dish).
  9. Remove dead and unfertilized eggs.
    NOTE: Healthy fertilized eggs are translucent and round. Remove cloudy, white, unfertilized, or deformed eggs.
  10. Double-check the fish to avoid mixing sexes or cross-contaminating transgenic tanks.
  11. Return the males and females to their respective tanks.
  12. Place the embryos in a 28.5°C incubator overnight. Do not exceed the embryo density specified in Step 1.8.
  13. Bleach the embryos on the following morning to remove contaminants.
    1. Prepare a working bleach solution consisting of 100 µL bleach in 200 mL system water. Prepare a neutralization solution consisting of 0.5 g sodium thiosulfate in 150–200 mL system water.
    2. Sort and transfer viable embryos into a mesh-net well insert. Submerge the insert in the bleach solution for 5 min.
    3. Transfer the insert directly into the sodium thiosulfate solution. Incubate for 2 min to neutralize residual bleach.
    4. Rinse the embryos in a final bath of fresh system water. Use a squirt bottle to gently transfer the embryos into clean petri dishes containing fresh system water.
  14. Return the embryos to the incubator after bleaching.
  15. Transfer larvae at 5–7 days post-fertilization (dpf) or later to a larger tank containing 5 ppt rotifer water for rearing.
    1. Prepare 5 ppt rotifer water by mixing 1 part stock rotifer water (15 ppt) with 2 parts zebrafish system water.
    2. Verify that the final salinity is 5 ppt using a conductivity meter before transferring the larvae.
      NOTE: Sorting EGFP+ MYCN larvae at 3–5 dpf facilitates identification of tumor-prone fish before rearing. Sorting MYCN embryos at this stage avoids false-negative identification because EGFP expression in neural crest cells may decrease later in development due to MYCN-induced apoptosis.

2. Screening Zebrafish for Fluorescent Markers

  1. Prepare a 0.4% (w/v) tricaine stock solution using system water.
  2. Transfer the fish to a clean petri dish containing only enough system water to cover the bottom.
  3. Add the tricaine stock solution to achieve a final concentration of approximately 0.005% (50 mg/L) for fish younger than 14 dpf or 0.012% (120 mg/L) for fish older than 14 dpf. Confirm adequate anesthesia by observing little to no voluntary movement.
  4. Remove excess water from the dish once the fish are anesthetized and no longer capable of swimming while maintaining visible opercular or cardiac movement.
  5. Screen the fish for fluorescence. Identify green or red fluorescence in the expected region (e.g., the interrenal gland region in MYCN fish).
  6. Separate the fish into groups using a stiff-bristled sorting tool.
    NOTE: Sort tumor-prone fish as early as possible because MYCN-induced apoptosis during later developmental stages may result in false-negative identification. Sort immune-reporter fish immediately before imaging at the larval stage or between 2 and 4 months of age for adult studies. This approach minimizes handling-associated stress and maximizes survival.
  7. Transfer positive and negative fish to separate tanks containing system water for continued rearing.
    NOTE: Step 3 describes handling and imaging of larvae younger than 21 dpf. Steps 4–6 describe fixation, tissue preparation, imaging, and image analysis of older fish.

3. Larval Live-Mounting and Imaging

  1. Place sorted larvae in a 100 mm petri dish containing tricaine fish water (see Step 2.3 for age-appropriate concentrations).
  2. Transfer the sedated larvae to a glass-bottom imaging dish.
  3. Use only enough solution to cover the larva and maintain sedation.
  4. Prepare 1%–1.5% low-melting agarose in fish water. Heat the agarose solution until fully dissolved.
  5. Allow the agarose to cool for 1–2 min. Maintain the agarose at 30°C using a heat block to prevent solidification prior to mounting.
  6. Position each larva laterally in the imaging dish.
  7. Apply approximately 30–200 µL of agarose over the larva to immobilize it. Use larger volumes for larger larvae and smaller volumes for smaller larvae.
  8. Once the agarose has solidified, add approximately 100 µL of 0.005% tricaine solution over the agarose.
  9. Image the larvae using a confocal microscope.
    1. Use a standard laser-scanning confocal microscope with a resonant scanner. Use a 40× objective with a numerical aperture greater than 1.10.
    2. Set the pinhole to 1.0 Airy unit (AU) for all imaging channels.
    3. Use a 488 nm laser for EGFP excitation and a 594 nm laser for mCherry excitation. Keep laser output below 25 mW to minimize photobleaching and phototoxicity. Adjust detector gain and laser power using a range indicator lookup table (LUT) to avoid pixel saturation and black clipping. Do not exceed a detector gain of 800 V because higher voltages introduce excessive noise.
    4. Use a voxel size of 0.42 × 0.42 × 6.0 µm (x, y, z) at a resolution of 1024 × 1024 pixels and a Z-step interval of 6.0 µm. Acquire optical sections continuously through the full volume of interest, typically spanning 90–120 µm in total imaging depth. Use highly sensitive photodetectors operating in 16-bit acquisition mode.
    5. Acquire images using unidirectional line scanning at a resonant scan frequency of 33.3 kHz (approximately 1.58 µs pixel dwell time). Use 2× or 4× averaging to reduce motion artifacts while limiting laser exposure.
      NOTE: Optimize tricaine exposure to maintain larval survival during imaging. After imaging, add fish water until the imaging dish is approximately half full.
  10. Add fresh fish water to the imaging dish immediately after imaging.
  11. Gently remove the larva from the agarose using a stiff-bristled stick and transfer the larva to a dish containing clean fish water for recovery.
  12. Return the fish to its original tank once normal swimming behavior resumes.
    NOTE: For fish older than 21 dpf, the skin overlying the tumor must be removed by cryosectioning to enable high-resolution confocal imaging.

4. Fish Fixation and Cryosectioning

  1. Euthanize fish in accordance with approved institutional animal care guidelines.
  2. Make a ventral incision extending from the anal pore to the pectoral girdle to expose the abdominal cavity and facilitate fixative penetration into internal tissues.
  3. Transfer the fish using forceps into a 1.5 mL tube containing 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) or a volume of fixative at least 10-fold greater than the tissue volume.
    NOTE: Avoid handling the region containing the tumor to preserve tissue integrity.
  4. Place the samples on a nutating rocker set to gentle continuous agitation (30 rpm) and incubate overnight for at least 12 h at 4°C.
    CAUTION: PFA is hazardous, toxic, and potentially carcinogenic. Wear appropriate personal protective equipment, including gloves and eye protection. Perform all procedures involving PFA in a fume hood and avoid inhalation of vapors.
  5. Remove the PFA and wash the samples at least four times with 1.5 mL of 1× PBS. Gently invert the tube two to three times during each wash.
  6. Transfer the fish to 30% sucrose prepared in PBS.
  7. Incubate the fish in a cold room overnight until the specimen sinks to the bottom of an upright tube (approximately 12 h), indicating adequate sucrose penetration.
  8. Remove excess sucrose solution by gently blotting the fish on a laboratory tissue.
  9. Transfer the fish to storage tubes.
  10. Store the samples at −80°C until imaging.
    NOTE: Process samples within 3 months to minimize fluorescent protein degradation and signal loss.
  11. Transport the fixed fish to the cryostat room on dry ice.
  12. Place the fish flat in a plastic mold with one sagittal side facing downward.
  13. Add Optimal Cutting Temperature (OCT) compound until the fish is fully covered.
  14. Freeze the mold at −20°C or in the cryostat until the OCT turns white.
    NOTE: Position the fish as flat as possible before freezing to facilitate consistent sectioning.
  15. Cryosection the zebrafish in the sagittal plane to remove the skin for subsequent whole-mount imaging.
    1. Cool the cryostat chuck.
    2. Apply room-temperature OCT to the chuck.
    3. Press the OCT fish block firmly into the OCT layer.
    4. Freeze the chuck and block until the newly applied OCT turns white.
    5. Mount the chuck in the cryostat.
    6. Cryosection the block at a thickness of 14 µm until the blade reaches the specimen.
      NOTE: Specimen shifting may occur during freezing, causing the fish to sit higher in the block than expected. Entry into the specimen is indicated by the appearance of translucent tissue replacing the opaque OCT background.
    7. Reduce the section thickness to 7 µm.
    8. Continue sectioning for one additional cut.
      NOTE: This procedure removes the skin while preserving a uniform sagittal profile across the specimen.
    9. Confirm the endpoint by identifying skin in the collected sections.
      ​NOTE: Skin appearance in sections provides a more reliable indicator than eye morphology. Perform this procedure only when the skin obscures imaging of the tumor microenvironment.
  16. Warm the chuck gently with your hands.
  17. Remove the OCT block from the chuck using forceps. Avoid disturbing the cut surface of the specimen.
  18. Trim excess OCT surrounding the fish while the OCT remains frozen.
  19. Remove the fish from the OCT block using forceps once the OCT softens to a viscous consistency.

5. Mounting and Imaging of Fish with Skin Removed

  1. Place the fish in a glass-bottom dish with the sectioned sagittal side facing the glass surface.
    NOTE: Maintain the orientation of the fish during handling. Identifying the sectioned side may become difficult after the fish is removed from the OCT block.
  2. Prepare 1%–1.5% low-melting agarose.
  3. Apply approximately 30–50 µL of agarose to the dorsal and ventral sides of the fish to secure the sample.
    NOTE: Avoid placing agarose beneath the fish because it can reduce image quality. Use only the minimum amount of agarose required for stabilization.
  4. Keep the sample on ice during transport. Image the sample within 2 h of mounting.
  5. Transport the sample to the confocal microscopy facility.
  6. Image the sample using confocal microscopy.
    1. Use a standard laser-scanning confocal microscope with a resonant scanner. A two-photon microscope may be used when autofluorescence limits image quality. Use a corrected 20× objective lens with a numerical aperture of at least 0.80.
    2. Set the pinhole to 1.0 Airy unit (AU) for all imaging channels.
    3. Use a 488 nm laser for EGFP excitation and a 594 nm laser for mCherry excitation. Keep laser output below 25 mW. Adjust detector gain and laser power using a range indicator lookup table (LUT) to avoid pixel saturation and black clipping. Do not exceed a detector gain of 800 V because higher voltages introduce excessive noise.
    4. Configure image acquisition parameters as follows.
      1. Use a voxel size of 0.42 × 0.42 × 6.0 µm (x, y, z) at a resolution of 1024 × 1024 pixels and a Z-step interval of 6.0 µm.
      2. Acquire spectral image stacks through the full volume of interest, typically spanning a total Z-stack acquisition range of 90–120 µm (15–20 optical sections).
      3. Initiate image acquisition in the fluid space immediately above the tissue surface and continue through the full target imaging depth to ensure complete coverage of the specimen.
      4. Acquire images using highly sensitive photodetectors operating in 16-bit acquisition mode.
    5. Acquire images using unidirectional line scanning at a resonant scan frequency of 33.3 kHz (approximately 1.58 µs pixel dwell time) with 8× line averaging.

6. Image Analysis and Quantification

  1. Image visualization using Fiji (version 1.54p).
    1. Open the confocal z-stack images.
    2. Merge colors to see channel overlay by selecting Image > Color > Merge Channels.
    3. Generate a projection of the full z-range containing the imaging region by selecting Image > Stacks > Z Project. Enter the slice range of interest, as well as Max Intensity projection type. This will display the 2D projection of the image.
      NOTE: For larval fish, it is feasible to capture the whole TME by confocal microscopy. Therefore, we recommend the volumetric normalization and quantification method for the TME analysis. For older fish, it is impossible to capture the whole TME by confocal microscopy; therefore, we recommend applying the area-based normalization and quantification method. Based on our analysis and quantification of the larval fish, both methods can provide reliable biological results.
  2. Volumetric normalization and quantification of the TME in the larval fish.
    1. Quantifying tumor volume using MATLAB R2022b (download the package from the Matlab website) and Bio-Formats package (download from the openmicroscopy website).
    2. The user runs a script that performs the following tasks:
      1. Prompt the user to select a .CZI file from their computer.
      2. Read the image and extract the data from the channel containing the tumor region.
      3. Identify the region for each slice that is the tumor region through a threshold of fluorescence (can be through user input or machine learning), keeping the same threshold for all analyzed images for consistency.
      4. Outline the tumor region and display the image with the tumor region outlined in a collage of all slices for user verification.
      5. Calculate the area of the tumor region per slice in pixels and convert to mm2 using the scale of the image and imaging settings.
      6. Multiply the area of the tumor region by the depth (stored in the file’s metadata) to get the volume of each slice.
      7. Sum the calculated values of each slice to obtain the total volume of the imaged tumor and its surrounding region in voxels or mm3.
      8. Scale both volume measurements by multiplying by 104 for tumor volume and 106 for the surrounding region. This will create a more reasonable cell number:volume ratio during normalization.
    3. Quantifying cell number.
      1. Open the confocal z-stack images.
      2. Merge colors to see channel overlay by selecting Image > Color > Merge Channels.
      3. Generate a 3D view of the full z-range containing the imaging region by selecting Image > Stacks > 3D Project. Select Brightest Point as the projection method and other settings to the desired values.
      4. Due to the small number of cells, manually count and tabulate the cells, separating cells located within the tumor boundary from those located outside the tumor boundary.
        NOTE: Cells on the boundary of the tumor region are counted as being outside.
    4. Normalization.
      1. For infiltrating cells inside the tumor region, divide the number of cells by the scaled tumor volume. For the cells outside the tumor region, divide the number of cells outside the tumor volume by the scaled volume of the full image minus the tumor volume.
      2. Generate a table that includes the following headings: “fish identification”, “tumor volume”, “# of cells inside tumor region”, “# of cells outside tumor region”, “normalized cells inside”, “normalized cells outside”.
  3. Area-based normalization and quantification of the TME.
    1. Define the tumor region of interest (ROI) using the Rectangle or Freehand selection tool to outline the tumor.
    2. Add the ROI to the ROI Manager by pressing T or by selecting Analyze > Tools > ROI Manager > Add.
    3. Measure the ROI area using Analyze > Measure.
    4. Confirm that Area is selected under Set Measurements before recording measurements.
    5. Save the ROI in the ROI Manager.
    6. Apply the same ROI to all images using ROI Manager > More > Multi-Measure.
    7. Do not resize the ROI between images.
    8. Manually count cells within the ROI. If cells are more, count cells using the Cell Counter plugin by selecting Plugins > Analyze > Cell Counter.
    9. Mark each counted cell by left-clicking when its centroid falls within the ROI.
    10. Scroll through the z-stack to verify cell identity.
    11. Avoid counting the same cell more than once across multiple image planes.
    12. Count infiltrating cells separately if they contact or lie within the tumor boundary.
    13. Record infiltrating cells as a distinct category even when the cell centroid falls outside the ROI.
    14. Convert the ROI area from pixels to mm2.
    15. Obtain the pixel width, pixel height, and Z-step interval from Image > Properties.
    16. Calculation of normalized cell counts.
      1. Divide the raw cell count for each fish by the corresponding ROI area value.
      2. Record the resulting value as the normalized cell count.
      3. For absolute density measurements, calculate the physical area using the image calibration values obtained from Image > Properties.
      4. Divide the raw cell count by the calculated area (mm2).
      5. Record the result as cell density expressed as cells/mm2.

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Risultati

Il flusso di lavoro sperimentale complessivo è mostrato nella Figura 1. Zebrafish adulti Tg(cd4-1:mCherry) sono stati incrociati con zebrafish Tg(dβh:MYCN;dβh:EGFP) per generare prole transgenica doppia in cui le cellule CD4+ e le cellule esprimenti dβh sono state etichettate in modo differenziale mediante fluorescenza mCherry ed EGFP, rispettivamente. Gli embrioni sono stati selezionati in base ai marcatori fluorescenti appropriati e successivamente ana...

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Discussione

Il protocollo descritto in questo articolo utilizza il pesce zebra come modello in vivo per visualizzare e quantificare le cellule immunitarie all'interno del microambiente tumorale (TME). Diversi fattori sono fondamentali per l'attuazione corretta di questo approccio. In primo luogo, è essenziale una selezione e una validazione adeguate di linee di pesci zebra transgenici composti. I larvi devono essere sottoposti a screening per verificare i pattern attesi di espressione fluorescente e, se necessario, conferma...

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Dichiarazioni

Gli autori dichiarano di non avere interessi concorrenti.

Ringraziamenti

Ringraziamo per il sostegno fornito dagli Istituti Nazionali di Sanità (NIH; 1UL1TR001430, CA215059 e NS140967), dalla American Cancer Society (RSG-17-204-01-TBG), dalla National Science Foundation (1911253) e dalla Alex’s Lemonade Stand Foundation a H.F.; e da una Warren Alpert Distinguished Scholars Fellowship a X.Q. Y.W. ha ricevuto sostegno dal Boston University Undergraduate Research Opportunities Program (UROP) e dal programma della borsa estiva St. Baldrick’s. M.V. è stato sostenuto dal programma della borsa estiva St. Baldrick’s. Il contenuto di questo articolo è esclusiva responsabilità degli autori e non rappresenta necessariamente le opinioni ufficiali del NIH.

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Materiali

Elenco dei materiali utilizzati in questo articolo
NomeAziendaNumero di catalogoCommenti
200 µL Gel Loading TipMilliporeSigma1022-0600Utilizzato per la manipolazione delle larve
Piante ArtificialiThe Hidden Reef62874200000000000Arricchimento riproduttivo opzionale
Ammoniaca ClorataFisher Scientific50371500Utilizzata per la decontaminazione degli embrioni
Plugin Contatore CelluleNational Institutes of Health (NIH)N/APlugin Fiji utilizzato per la quantificazione cellulare
Computer e MonitorHP2TB68A8#ABAUtilizzati per l'acquisizione e l'analisi delle immagini
Microscopio ConfocaleZeissLSM 710Utilizzato per l'imaging di larve e adulti
Cassetta per Criomodellatura, in PlasticaEpredia22-050-160Utilizzata per l'inclusione in OCT
CriostatoThermo Fisher Scientific95-664-0EC70Utilizzato per la criosezionatura
Ghiaccio SeccoFisher ScientificN/AUtilizzato per il trasporto dei campioni
Software FijiNational Institutes of Health (NIH)Versione 1.54pSoftware per l'analisi delle immagini
Rete a Maglia FineCarolina Biological651345Utilizzata per la raccolta degli embrioni
Incubatrice per PesciVWR35960-056Mantenuta a 28.5°C
PinzeFisher Scientific10-316BUtilizzate per la manipolazione dei campioni
Piatto con Fondo in VetroIBIDI81218-200Utilizzato per l'imaging
Agarosio a Punto di Fusione BassoProlab Supply9012-36-6Utilizzato per il montaggio di larve e adulti
Acquario per Accoppiamento (serbatoio principale, divisore, sezione a griglia)Carolina Biological161937Utilizzato per la riproduzione del pesce zebra
Agitatore RotanteMidsciR2D-30Utilizzato per la fissazione durante l'incubazione notturna
Composto OCTTissue-Tek4583Mezzo di inclusione per criosezionatura
Paraformaldeide (PFA)Sigma Aldrich158127-500GFissativo
Piatto di Petri, in PlasticaMilliporeSigmaBR452000Utilizzato per la manipolazione di embrioni e larve
Soluzione Salina Fosfato-Tampone (PBS)Gibco14080-055Utilizzata per lavaggi e procedure di fissazione
Pipetta di Trasferimento in PlasticaFisher Scientific50-998-100Utilizzata per il trasferimento di embrioni e larve
Lama di RasoiFisher Scientific12-640Utilizzata durante la preparazione dei campioni
Misuratore di ConduttivitàYSIEC300ACC-04Utilizzato per verificare la salinità dell'acqua per rotiferi
RotiferiThe Hidden Reef116151603Utilizzati per l'allevamento delle larve
Tiosolfato di SodioThermo Fisher ScientificAAA1762936Utilizzato per la neutralizzazione dell'ammoniaca clorata
SaccarosioFisher ScientificS25590AReagente crioprotettivo
Tricaina MetanosolfonatoWestern ChemicalNC0872873Anestetico; venduto da Fisher Scientific
PinzetteFisher Scientific12-000-122Utilizzate per la manipolazione dei campioni
Microscopio Fluorescenza UprightLeicaM165Utilizzato per la selezione mediante fluorescenza

Riferimenti

  1. Anderson NM, Simon MC. The tumor microenvironment. Curr Biol. 2020;30(16):R921-R925.
  2. Kumagai S, Momoi Y, Nishikawa H. Immunogenomic cancer evolution: A framework to understand cancer immunosuppression. Sci Immunol. 2025;10(105):eabo5570.
  3. Keren L, et al. A structured tumor-immune microenvironment in triple negative breast cancer revealed by multiplexed ion beam imaging. Cell. 2018;174(6):1373-1387.e19.
  4. Gerner MY, et al. Histo-cytometry: A method for highly multiplex quantitative tissue imaging analysis applied to dendritic cell subset microanatomy in lymph nodes. Immunity. 2012;37(2):364-376.
  5. Pittet MJ, Weissleder R. Intravital imaging. Cell. 2011;147(5):983-991.
  6. Wienke J, et al. The immune landscape of neuroblastoma: Challenges and opportunities for novel therapeutic strategies in pediatric oncology. Eur J Cancer. 2021;144:123-150.
  7. Spitzer MH, Nolan GP. Mass cytometry: Single cells, many features. Cell. 2016;165(4):780-791.
  8. Roy D, et al. Zebrafish—A suitable model for rapid translation of effective therapies for pediatric cancers. Cancers (Basel). 2024;16(7):1361.
  9. White RM, et al. Transparent adult zebrafish as a tool for in vivo transplantation analysis. Cell Stem Cell. 2008;2(2):183-189.
  10. MacRae CA, Peterson RT. Zebrafish as tools for drug discovery. Nat Rev Drug Discov. 2015;14(10):721-731.
  11. Miao KZ, et al. Tipping the scales with zebrafish to understand adaptive tumor immunity. Front Cell Dev Biol. 2021;9:660969.
  12. Povoa V, et al. Innate immune evasion revealed in a colorectal zebrafish xenograft model. Nat Commun. 2021;12(1):1156.
  13. Wang J, et al. Novel mechanism of macrophage-mediated metastasis revealed in a zebrafish model of tumor development. Cancer Res. 2015;75(2):306-315.
  14. Loveless R, Shay C, Teng Y. Unveiling tumor microenvironment interactions using zebrafish models. Front Mol Biosci. 2020;7:611847.
  15. Dee CT, et al. CD4-transgenic zebrafish reveal tissue-resident Th2- and regulatory T cell-like populations and diverse mononuclear phagocytes. J Immunol. 2016;197(9):3520-3530.
  16. Murali Shankar N, et al. Preclinical assessment of CAR-NK cell-mediated killing efficacy and pharmacokinetics in a rapid zebrafish xenograft model of metastatic breast cancer. Front Immunol. 2023;14:1254821.
  17. Tan KE, et al. LMO1 expression in neuroblastoma cells reprograms tumor-associated macrophages to promote metastasis. iScience. 2026;29(3):115144.
  18. Fior R, et al. Single-cell functional and chemosensitive profiling of combinatorial colorectal therapy in zebrafish xenografts. Proc Natl Acad Sci U S A. 2017;114(39):E8234-E8243.
  19. Fazio M, et al. Zebrafish patient avatars in cancer biology and precision cancer therapy. Nat Rev Cancer. 2020;20(5):263-273.
  20. Qin X, et al. CKLF instigates a “cold” microenvironment to promote MYCN-mediated tumor aggressiveness. Sci Adv. 2024;10(11):eadh9547.
  21. Ludin A, et al. CRATER tumor niches facilitate CD8+ T cell engagement and correspond with immunotherapy success. Cell. 2025;188(24):6720-6736.e26.
  22. Zhu S, et al. LMO1 synergizes with MYCN to promote neuroblastoma initiation and metastasis. Cancer Cell. 2017;32(3):310-323.e5.
  23. Tao T, et al. The pre-rRNA processing factor DEF is rate limiting for the pathogenesis of MYCN-driven neuroblastoma. Oncogene. 2017;36(27):3852-3867.
  24. Zimmerman MW, et al. MYC drives a subset of high-risk pediatric neuroblastomas and is activated through mechanisms including enhancer hijacking and focal enhancer amplification. Cancer Discov. 2018;8(3):320-335.
  25. Dankert EN, Look AT, Zhu S. Unraveling neuroblastoma pathogenesis with the zebrafish. Cell Cycle. 2018;17(4):395-396.
  26. Langenau DM, et al. In vivo tracking of T cell development, ablation, and engraftment in transgenic zebrafish. Proc Natl Acad Sci U S A. 2004;101(19):7369-7374.
  27. Lam SH, et al. Development and maturation of the immune system in zebrafish, Danio rerio: A gene expression profiling, in situ hybridization and immunological study. Dev Comp Immunol. 2004;28(1):9-28.
  28. Zhen A, et al. CD4 ligation on human blood monocytes triggers macrophage differentiation and enhances HIV infection. J Virol. 2014;88(17):9934-9946.
  29. Ferrero G, et al. The macrophage-expressed gene (mpeg)1 identifies a subpopulation of B cells in the adult zebrafish. J Leukoc Biol. 2020;107(3):431-443.
  30. Hall C, et al. The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish. BMC Dev Biol. 2007;7:42.
  31. Lorenzini F, et al. Melanoma innervation, noradrenaline and cancer progression in zebrafish xenograft model. Cell Death Discov. 2025;11(1):260.
  32. Ramakrishnan G, et al. Real-time imaging reveals a role for macrophage protrusive motility in melanoma invasion. J Cell Biol. 2025;224(2):e202403096.

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