Intracranial Breast Cancer Brain Metastases Mouse Model for Immune and Stromal Cells Characterization in Meninges and Olfactory Bulb

Summary

The protocol establishes breast cancer brain metastases via intracranial inoculation and to characterize by flow cytometry key lymphatic and immune cell subsets that regulate brain immunity. These analyses provide insights into the immune and stromal cell dynamics that shape the brain microenvironment, informing the development of new therapeutic strategies.

Abstract

The central nervous system (CNS) is surrounded by specialized immune interfaces, including the meninges and the olfactory bulb, which can serve both as permissive routes for metastatic colonization and as entry points for anti-tumor immune responses. Understanding how immune cell dynamics evolve within these structures during metastatic progression is essential for identifying mechanisms that either support or restrict tumor growth in the brain. This protocol details the establishment of a breast cancer brain metastases (BCBM) mouse model through intracranial injection of E0771 breast cancer cells into C57BL/6 mice. After tumor development, the meninges and olfactory bulb are carefully dissected, enzymatically digested, and processed into single-cell suspensions. These populations are then analyzed using multiparametric spectral flow cytometry to comprehensively characterize lymphoid, myeloid, and stromal cell subsets within CNS-associated immune niches. This workflow enables comparative analyses between tumor-bearing and non-tumor controls, identifying alterations within CNS immune interfaces that may underlie tumor immune evasion and represent potential therapeutic targets. Overall, the protocol provides a reproducible approach to study BCBM and interrogate immune regulation at CNS-peripheral immune system interfaces. It can be adapted for additional cancer cell lines, immunotherapies, or genetic mouse models to investigate immune resistance mechanisms and therapeutic response within the brain microenvironment.

Introduction

Brain metastases are the most common malignant tumors affecting the central nervous system (CNS), occurring more frequently than primary brain tumors. Lung cancer, breast cancer, and melanoma are the primary cancers that most often metastasize to the brain¹. Among these, breast cancer brain metastases (BCBM) represent a major clinical challenge, affecting 10%–30% of patients with metastatic disease and showing increasing incidence due to improved systemic therapies and advances in neuroimaging². The prognosis for patients with BCBM remains poor, as available treatments – including surgery, radiotherapy, and chemotherapy – are largely palliative, and neurological symptoms severely impact quality of life³. Although targeted therapies and immunotherapies are being explored as complementary strategies, the clinical benefit of immunotherapy in brain metastases remains limited^4,5,6.

Recent work has refined our understanding of brain immunity and renewed interest in functional lymphatic pathways associated with the CNS, including lymphatic vessels in the meninges⁷ and nasal mucosa⁸. These structures form a continuous lymphatic network surrounding the brain and extending adjacent to the olfactory bulb and the cribriform plate, ultimately connecting to cervical lymph nodes. This system plays key roles in fluid homeostasis, immune surveillance, and immune cell trafficking between the CNS and peripheral lymphoid organs⁹. As interest grows in immunotherapeutic approaches for brain tumors and metastases, these lymphatic-immune interfaces are emerging as promising, yet still understudied, routes through which systemic immunity may communicate with intracranial lesions. In addition, the mechanisms governing immune cell recruitment into the brain tumor microenvironment and how these cells interact within this unique environment remain poorly understood. Together, these gaps underscore the need for preclinical models and procedures that allow comprehensive analysis of tumor-immune interactions and support the development of strategies to improve anti-tumor immunity against brain metastases.

Here, we describe a protocol that combines the generation of a reproducible, immunocompetent BCBM mouse model through stereotaxic intracranial inoculation of E0771 cells with the isolation and dissociation of the meninges and olfactory bulb for immunophenotyping by spectral flow cytometry. This approach enables controlled modeling of BCBM to assess the safety and efficacy of therapeutic interventions and allows in-depth characterization of immune and stromal populations at the brain lymphatic-immune interface, providing insight into the cellular players that shape the brain tumor microenvironment and influence treatment responses.

Animal models remain indispensable for studying brain metastases, as many critical aspects of metastatic progression cannot be replicated in vitro. Several in vivo mouse models have been developed to generate brain metastases, each with specific advantages and limitations¹⁰. Spontaneous metastasis models, including orthotopic implantation or genetically engineered mouse models, more closely mimic clinical disease progression but rarely generate intracranial lesions, require long experimental timelines, and often yield high variability due to widespread extracranial disease. Systemic approaches, including intracardiac, intracarotid, or tail-vein injections, introduce tumor cells into the circulation and better mimic hematogenous dissemination, blood-brain barrier extravasation, and colonization. However, these models do not capture the early steps of primary tumor invasion and frequently produce extensive and variable extracranial metastases, particularly in the lungs. Among systemic models, intracarotid injection has the lowest variability, but it requires advanced surgical expertise. Alternatively, intracranial injection offers several advantages, including precise control over tumor location, rapid and reproducible tumor development, and reduced variability between experiments. This approach may also be complemented by orthotopic primary tumor implantation followed by surgical resection to more accurately mimic the metastatic course. Although intracranial injection does not recapitulate the full metastatic cascade and is not suited for studying early invasion and dissemination, it provides a robust model to evaluate therapeutic efficacy against established lesions and to investigate interactions within the brain microenvironment. A known limitation is that the injection itself may induce local neuroinflammation, potentially influencing immune readouts, which can be assessed by comparing immune profiles in injected and non-injected mice.

The immune characterization of the meninges and olfactory bulb remains a relatively new and underexplored area of research. Most studies rely on whole-brain or cervical lymph node samples¹¹, which fail to capture the distinct cellular composition and specialized immune functions of these lymphatic-associated tissues. Recovering both structures provides a broader and more informative view of brain immunity. When combined with multiparametric spectral flow cytometry, this workflow supports high-throughput profiling of abundant and rare immune cell populations involved in CNS-peripheral immune communication. This includes quantification of surface and intracellular markers to identify cell subsets, assess activation states, and evaluate treatment-induced changes. Other methodologies for CNS immune analysis not covered by this protocol include immunohistochemistry (IHC) and immunofluorescence (IF), which preserve the spatial distribution of immune cells but are limited by low marker multiplexing, subjective quantification, and underrepresentation of rare cell types¹². More recently, single-cell RNA sequencing (scRNA-seq) has expanded the ability to profile CNS immune populations by enabling the discovery of cellular states and transcriptional programs at high resolution. Despite offering powerful transcriptomic insight, scRNA-seq is limited by cost, dissociation artifacts, transcript dropout, and loss of protein-level resolution^13,14. Flow cytometry, while lacking spatial analysis and being sensitive to dissociation-related effects, offers superior phenotypic resolution through an advanced multiparametric panel and provides robust quantitative immune profiling.

Together, the methodologies described here provide a flexible and powerful platform for modeling BCBM and interrogating immune dynamics at CNS lymphatic interfaces, supporting preclinical development of therapies targeting both the brain tumor microenvironment and its associated immune regulatory pathways.

Protocol

All animal procedures were conducted in accordance with the guidelines of Faculty of Pharmacy, University of Lisbon. The protocols were reviewed and approved by the Portuguese competent authority for animal protection, Direção-Geral de Alimentação e Veterinária (Reference 0421/000/000/2021).

1. Preparation of E0771 cells for in vivo implantation

1. In a sterile hood, culture E0771 cells in a T75 flask using Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (v/v) Fetal bovine serum (FBS) and 1% (v/v) penicillin and streptomycin (PEST).
   1. Ensure that cells are authenticated by STR profiling upon receipt from the supplier and confirmed mycoplasma-free by routine testing. Culture tumor cells for at least 2–8 passages post-thawing prior to brain implantation.
2. On the day of implantation, ensure the cells are healthy and 80-90% confluent. Remove medium and wash cells with phosphate-buffered saline (PBS).
3. Add trypsin to the flask and incubate for 5 min at 37 °C. Then, add three times the volume of supplemented medium (DMEM with 10% v/v FBS and 1% v/v PEST) to neutralize trypsin and dissociate cells.
4. Transfer cells to a tube and centrifuge at 300 x g for 5 min at room temperature (RT). Remove supernatant and resuspend the pellet in PBS for cell counting.
5. Centrifuge again (300 x g for 5 min), discard supernatant, and resuspend cells in PBS at a concentration of 1 x 10⁵ E0771 cells in 2 μL per animal. Keep the cell suspension on ice until tumor inoculation. Prepare the cell suspension immediately before injection.

2. Intracranial tumor inoculation using a stereotaxic apparatus

1. Obtain female C57BL/6 mice (6-8 weeks old) and acclimate them to the animal facility for at least 7 days prior to surgery. Keep house mice under controlled conditions with a 12-h light/ dark cycle and provide food and water ad libitum.
   NOTE: A minimum of five animals per experimental group is recommended to ensure sufficient statistical power for meaningful comparative immune profiling between tumor-bearing and control conditions.
2. Prepare the surgical field and all required materials before starting the procedure:
   1. Surgical instruments: scalpel blades, scissors, forceps, needle holders, suture wire.
   2. Equipment and others: stereotaxic instrument, glass syringe, drill, shaver, cotton swabs, heating pad.
   3. Anesthesia: Prepare ketamine-xylazine (K-X) (25 mg/mL and 2 mg/mL, respectively) in PBS in a 15 mL tube.
   4. Analgesia: Prepare buprenorphine at 15 μg/mL in PBS in a 15 mL tube.
   5. Other solutions: povidone-iodine, hydrogen peroxide 3% v/v (H₂O₂), ophthalmic ointment.
      CAUTION: H₂O₂ is harmful if swallowed and can cause skin irritation.
3. Anesthetize the mouse by intraperitoneal (i.p.) injection of K-X (100 μL per 10 g body weight). Confirm adequate anesthesia by the absence of response to toe pinch.
4. Apply ophthalmic ointment to both eyes to prevent dehydration and place the mouse on a heating pad throughout the procedure.
5. Shave the scalp and disinfect the surgical area with povidone-iodine. Make a 1 cm longitudinal incision along the midline to expose the skull.
6. Clean the exposed skull with H₂O₂ using a cotton swab in a circular motion to visualize bregma. If excessive foam occurs, remove it with a dry swab.
7. Position the mouse in the stereotaxic frame. Set the bregma as the reference point (0.0) and measure 2.5 mm lateral and 0.5 mm anterior to the bregma. Mark the injection site.
8. Remove the mouse and drill a small hole at the marked coordinates. Avoid drilling too deeply to prevent brain damage.
9. Mount the syringe on the injector. Resuspend cells and load the glass syringe with enough volume for two injections (4–5 μL). Do not prepare more than two injections at a time to avoid cell settling.
10. Reposition the mouse in the stereotaxic, align the needle with the hole, and slowly lower it to a depth of 3.0 mm into the brain parenchyma. Inject cells (or sterile PBS for sham control) at 1 μL/min.
11. After injection, keep the needle in place for at least 1 min, retract 1.5 mm, hold for another minute, then withdraw completely. This minimizes backflow and ensures proper cell deposition.
12. Close the incision with sutures and apply povidone-iodine. Administer buprenorphine (100 μL) subcutaneously.
13. Keep the mouse on a heating pad until full recovery from anesthesia. Return the mouse to a clean cage and monitor daily for distress, neurological signs, or infection.

3. Mouse intracardiac perfusion

1. Anesthetize mice by i.p. injection of K-X (100 μL per 10 g body weight). Confirm adequate anesthesia by the absence of response to toe pinch.
2. Using scissors, make a 5–6 cm incision through the abdominal wall and two incisions through the ribcage up to the collarbone. Take care not to cut any major blood vessels.
3. With the heart exposed, cut the right atrium to allow blood flow. Inject ice-cold PBS into the left ventricle with a 20 mL syringe equipped with a 25G × 1 inch needle.
4. Stop perfusion when the fluid flowing out is clear of blood. For adult mice, it takes around 20 mL PBS.

4. Isolation of the mouse meninges and olfactory bulb

1. Following intracardiac perfusion, decapitate the mouse using surgical scissors. Remove the scalp completely to expose the skull.
2. Using scissors, cut the skull in the transverse plane from the posterior region toward the eyes, taking care not to damage the underlying brain tissue. Gently extract the brain using fine forceps, avoiding contact with or scraping of the inner skull surface.
3. After brain removal, the dural meninges remain attached to the inner skull surface. Submerge the skull in ice-cold PBS for at least 5 min to facilitate intact separation of the meninges.
4. Dissect the olfactory bulb from the anterior region of the brain and transfer it into ice-cold PBS for temporary storage until processing.
5. Using fine-tipped forceps, gently detach the meninges from the inner skull surface by carefully lifting and peeling the thin tissue layer. Transfer the meninges into ice-cold PBS until further processing. Avoid tearing the tissue to ensure optimal downstream cell recovery.

5. Tissue dissociation into single cells

1. Meninges
   1. Transfer meninges into 3 mL RPMI 1640 containing 0.05% collagenase II and DNase I. Incubate for 20 min at 37 °C, gently agitating every 10 min. Over-digestion reduces cell viability and should be avoided.
   2. Pass digested tissue through a 40 μm cell strainer into a 50 mL tube. Mechanically dissociate remaining tissue by gently pressing a syringe plunger against the strainer and wash with 5 mL PBS.
   3. Centrifuge at 300 x g for 5 min at 4 °C. Discard supernatant and resuspend pellet in 3 mL PBS.
   4. Repeat centrifugation. Discard supernatant, resuspend final pellet in remaining volume, and transfer to a U-bottom 96-well plate.
2. Olfactory bulb
   1. Remove PBS and mechanically dissociate tissue using a syringe plunger. Add 3 mL digestion solution containing 0.02% collagenase II in Hanks' balanced salt solution (HBSS). Incubate for 20 min at 37 °C, gently agitating every 10 min.
   2. Pass cell suspension through a 70 μm strainer into a 50 mL tube and wash with 10 mL PBS.
   3. Centrifuge at 300 x g for 5 min at 4 °C. Discard supernatant and resuspend final pellet in remaining volume. Transfer to a U-bottom 96-well plate.
3. Store the plates at 4 °C until staining.
   NOTE: Perform staining on the same day to ensure optimal cell viability.

6. Cell staining and flow cytometry acquisition

1. Panel design
   1. Please see Table 1.
      ​NOTE: The panel in this protocol was designed for a spectral flow cytometer equipped with 4 lasers (violet, blue, yellow-green, and red) and a total of 51 detectors (48 fluorescence + 3 scatter channels). The panel needs to be designed based on the configuration of the flow cytometer and the proteins/markers of interest.
2. Cell Staining
   1. Centrifuge the U-bottom 96-well plates at 300 x g for 5 min at 4 °C. Carefully discard supernatant. From this step onward, protect plates from light.
   2. Add 50 μL viability dye (FVD, dilution 1:5000 in PBS) to appropriate wells and incubate for 20 min at RT. Wash with 150 μL PBS, centrifuge at 300 x g for 5 min at 4 °C, and discard supernatant. Add viability dye only to viability controls and samples.
   3. Block Fc receptors by incubating cells with 30 μL of anti-CD16/CD32 antibody (0.01 μg/mL in FACS buffer: PBS with 2 mM EDTA and 0.5% BSA) for 10 min on ice. Wash with 170 μL FACS buffer, centrifuge at 300 x g for 5 min at 4 °C, and discard supernatant.
   4. Add 30 μL antibody mix or single-color controls (prepared according to the antibody dilutions listed in the Materials Table, in FACS buffer) and incubate for 20 min on ice. Wash with 170 μL FACS buffer, centrifuge at 300 x g for 5 min at 4 °C, and discard supernatant.
   5. Fix cells with 100 μL 2% paraformaldehyde (PFA) in PBS for 20 min at RT. Wash with 100 μL of PBS, centrifuge at 300 x g for 5 min at 4 °C, and discard supernatant.
      NOTE: For intracellular markers, replace this step with fixation and permeabilization using a commercial buffer set, then perform intracellular staining before final resuspension.
   6. Resuspend final cell pellets in 200 μL of FACS buffer. Store plates at 4 °C until acquisition on the flow cytometer.
3. Single-color-stained and Fluorescence Minus One (FMO) controls
   1. Single-color-stained samples are required for unmixing optimization. Cells are used for single-color-stained samples for the markers showing positive and negative peaks. Otherwise, the use of compensation beads is preferred and recommended to obtain optimal unmixing. Prepare compensation beads for fluorescent-conjugated antibodies according to the manufacturer’s protocol.
      NOTE: Preparing FMO controls is an important step to achieve proper gating, especially for the markers that do not show clear positive and negative peaks. Prepare multiple FMO samples for different fluorochromes as needed.
   2. Prepare FMO samples for cell surface antigens by following the steps in Part 6.2 but exclude the antibody of interest from the antibody mix at step 6.2.4.
4. Flow cytometry acquisition and analysis
   1. Turn ON the cytometer, launch the flow cytometer acquisition software, and prepare the quality-control (QC) beads (1 drop in 300 µL PBS) in a 12 x 75 mm sample tube while the machine is warming up (at least 30 min).
   2. Clean the instrument by running: 3 min 10 % v/v bleach, 30 min H₂O at flow rate high. Put the flow rate back to low for the next steps.
   3. Select the QC tab, load the tube and run the beads by pressing start. Once the QC is done, a message saying “Passed” pops up.
   4. Go to acquisition tab, select or create the experiment and start the acquisition by gating cells per FSC and SSC. Acquire unstained and single-color controls, run the unmixing wizard and save the settings. Proceed to acquire stained samples.

Results

Following intracranial inoculation of E0771 cells, mice developed a localized breast cancer lesion at the targeted stereotaxic coordinates. The stereotaxic coordinates targeting the striatal parenchyma (2.5 mm lateral, 0.5 mm anterior to bregma, and 3.0 mm ventral from bregma) were selected based on standard mouse brain atlases and previously described intracranial implantation approaches^15,16. This region is widely used for intracranial tumor implantation due to...

Discussion

By combining intracranial tumor implantation with the isolation, dissociation, and immune characterization of the meninges and olfactory bulb, this protocol enables detailed investigation of the dynamic interplay between the brain tumor microenvironment and CNS-associated immune interfaces. The method provides reproducible intracranial tumor formation, high-quality cell suspensions, and robust immune subset resolution. However, intracranial implantation bypasses the early stages of the metastatic cascade and does not ful...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alexa Fluor 594 anti-mouse CD45R/B220 antibody (clone RA3-6B2)	BioLegend	103254	used at 1:300 dilution
Alexa Fluor 700 anti-mouse CD8 antibody (clone 53-6.7)	BioLegend	100730	used at 1:50 dilution
APC anti-mouse P2RY12 antibody (clone S16007D)	BioLegend	848006	used at 1:80 dilution
Betadine	Selleck Biotechnology GmbH	S4678-100mg	Povidone-iodine
Bovine Serum Albumin (BSA), lyophilized powder, ≥96% (agarose gel electrophoresis)	Sigma-Aldrich	A2153	To prepare FACS Buffer
Brilliant Violet 421 anti-mouse Podoplanin antibody (clone 8.1.1)	BioLegend	127423	used at 1:80 dilution
Brilliant Violet 510 anti-mouse CD11b antibody (clone M1/70)	BioLegend	101263	used at 1:30 dilution
Brilliant Violet 570 anti-mouse NK1.1 antibody (clone PK136)	BioLegend	108733	used at 1:40 dilution
Brilliant Violet 650 anti-mouse CD19 antibody (clone 6D5)	BioLegend	115541	used at 1:50 dilution
Brilliant Violet 711 anti-mouse CD44 antibody (clone IM7)	BioLegend	103057	used at 1:80 dilution
Brilliant Violet 785 anti-mouse F4/80 antibody (clone BM8)	BioLegend	123141	used at 1:40 dilution
Bupaq 3 mg/mL	Bio5	1002241	Buprenorphine
Cell strainer 40-μm	Avantor	732-2757
Cell strainer 70-μm	Avantor	732-2758
Centrifuge 5910 Ri	Eppendorf	5943000011
Collagenase type II	Worthington Biochemical Corporation	LS004176
Corning Matrigel Growth Factor Reduced Basement Membrane Matrix	Corning	356231
Cytek Aurora Spectral Flow Cytometer	Cytek Biosciences
Dnase I	Worthington Biochemical Corporation	LS002006
Drill	Stoelting Europe	51630
Dulbecco's Modified Eagle Medium (DMEM), high-guclose, pyruvate	Gibco	41966029
E0771 cell line	ATCC	CRL-3436
Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA)	Merck	E4884	To prepare FACS Buffer
FCS Express Flow Cytometry Software	De Novo Software
Fetal Bovine Serum (FBS)	Gibco	A5256801
FITC anti-mouse CD3 (clone 17A2)	BioLegend	100204	used at 1:50 dilution
Ghost Dye Red 780	Cytek Biosciences	13-0865-T500	Viability dye (used at 1:5000 dilution)
HBSS, calcium, magnesium, no phenol red	Gibco	14025092
Heating Pad	MOBICLINIC
Hydrogen Peroxide 34.5 to 37% (H2O2)	ACROS	10002780
Ketamax 100 mg/mL	Bio3	1009495	Ketamine
Lubrithal Eye Gel	Bio4	1731329
N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) 1M	Gibco	15630080
Paraformaldehyde (PFA) 16 %	Avantor	43368	To prepare Fixation Buffer
Pacific Blue anti-mouse Ly-6G antibody (clone 1A8)	BioLegend	127612	used at 1:50 dilution
PE anti-mouse Ly-6C antibody (clone HK1.4)	BioLegend	128008	used at 1:300 dilution
PE anti-mouse P2RY12 antibody (clone S16007D)	BioLegend	848004	used at 1:80 dilution
PE/Cyanine5 anti-mouse CD11c antibody (clone N418)	BioLegend	117316	used at 1:80 dilution
PE/Cyanine7 anti-mouse CD80 antibody (clone 16-10A1)	BioLegend	104734	used at 1:100 dilution
PE-eFluor 610 anti-mouse CD31 antibody (clone 390)	eBioscience	61-0311-82	used at 1:160 dilution
PE/Fire 810 anti-mouse I-A/I-E (clone M5/114.15.2)	BioLegend	107667	used at 1:300 dilution
Penicillin-Streptomycin (PEST), 10 000 U/mL	Gibco	15140-122
PerCP/Cyanine5.5 anti-mouse CD45 antibody (clone 30-F11)	BioLegend	103132	used at 1:80 dilution
Phosphate-buffered saline (PBS) 10X, pH 7.4	Gibco	70011044
Prism (version 10)	GraphPad
RN Needle (27/51/3)S	Hamilton Company	7762-01
RPMI 1640 Medium, GlutaMAX Supplement	Gibco	61870044
Scalpel Blades, carbon steel	B Braun	BB510
Sedaxylan 20 mg/mL	Bio2	1112384	Xylazine
Spark Blue 574 anti-mouse CD4 antibody (clone GK1.5)	BioLegend	100490	used at 1:50 dilution
Stereotaxic Instrument	Stoelting Europe	51730D
Suture wire Silkam black 5/0 10x45 cm	B Braun	C0266639	non-absorbable
Surgical Tool Kit (Scissors, Clamps and Forceps, Clamps and Needle holders)	Stoelting Europe	52189
Syringes with needle 0.5 mL, 0.3x12 mm	B Braun	9151125
TruStain FcX PLUS anti-mouse CD16/32 antibody (clone S17011E)	BioLegend	156604	FcR block (used at 1:50 dilution)
Trypan Blue solution 0.4 %	Sigma-Aldrich	T8154
Trypsin-EDTA (0.25%), phenol red	Gibco	25200-072
U-bottom 96-well plate	Avantor	734-2328
50 mL tube	Avantor	525-0610
701 RN Microliter™ syringe	Hamilton Company	548-2273