Reprogramming of breast tumor–associated macrophages with modulation of arginine metabolism

Cell lines

A human monocytic cell line THP-1 (Cat. No. TIB-202), and SKBR3 (Cat. No. HTB-30) and BT-474 (Cat. No. HTB-20) cancer cell lines were purchased from the American Type Culture Collection. CA1d breast cancer cells were obtained from Karmanos Cancer Institute (Detroit, MI) under Material Transfer Agreement. PBMCs were obtained from STEMCELL Technologies and AllCells, LLC. Mouse HER2-positive NT2.5 cells derived from mammary tumors of MMTV-neu/FVB mice were kindly provided by Dr. Elizabeth M Jaffee at Johns Hopkins University under Material Transfer Agreement (57).

Cell culture and reagents

THP-1 cells were maintained at a density of 1 × 10⁶ cells/ml in RPMI 1640 medium (Cat. No. 11835055; Thermo Fisher Scientific) supplemented with 10% FBS, 1% penicillin–streptomycin, 2 mM GlutaMAX, 10 mM Hepes buffer, 45 g/liter glucose, and 1 mM sodium pyruvate (Cat. No. 15140122, Cat. No. 35050061, Cat. No. SH3023701, Cat. No. A2494001, and Cat. No. 11-360-070; Thermo Fisher Scientific). SKBR3 and BT-474 cells were cultured in McCoy’s 5A medium with 10% FBS and 1% penicillin–streptomycin, and CA1d cells were cultured in DMEM/F12 medium with 5% horse serum, 1% penicillin–streptomycin, hydrocortisone, cholera toxin, and insulin (Cat. No. H-0888, Cat. No. C8052-2MG, and Cat. No. I1882; Sigma-Aldrich, Inc.). NT2.5 cells were cultured in RPMI-1640 medium supplemented with 20% FBS, 1.2% Hepes, 1% GlutaMAX, 1% non-essential amino acids, 1% sodium pyruvate, 1% penicillin–streptomycin, and bovine insulin (10 mg/ml) (57). All the cells were maintained in a 37°C humidified incubator with 5% CO₂.

Modulation of arginine metabolism

For induction of NO, we used NO donors: S-nitroso-N-acetylpenicillamine (SNAP, 10 or 20 μM, Cat. No. N7892; Thermo Fisher Scientific); and S-nitrosoglutathione (GSNO, 100 or 200 μM, Cat. No. sc-200349B; Santa Cruz Bio), or SEP, a precursor of NOS cofactor tetrahydrobiopterin (20 or 100 μM; Career Henan Chemical Co). For NO inhibition, we used Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME, 2.5 mM, Cat. No. N5751-10G; Sigma-Aldrich); the NOS2 inhibitor: 1400W hydrochloride (100 μM, Cat. No. 81520; Cayman Chemical); or the NO scavenger: 4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO, 50 or 100 μM, Cat. No. ALX-430-001-M050; Enzo). For induction of PA, we treated cells with spermine (5 or 10 mM, Cat. No. 55513-100MG; Sigma-Aldrich). For inhibition of PA synthesis, we used the polyamine analog, N1,N11-diethylnorspermine tetrahydrochloride (DENSPM, 50 or 100 μM, Cat. No. 0468/10; Tocris). For inhibition of arginase 1 (Arg1), we used N-hydroxy-nor-L-arginine (nor-NOHA, 50 μM, Cat. No. 10006861; Cayman Chemical); for inhibition of ornithine decarboxylase 1, we used difluoromethylornithine (DFMO, 50 μM, Cat. No. D193-25MG; Sigma-Aldrich).

In vitro model of TAMs

PBMC-derived model

PBMCs were plated at a density of 1.5 × 10⁶ cells/ml in RPMI serum-free media and incubated for 3 h. Monocytes were isolated based on their adhesion to plastic. Serum-free media were aspirated to remove non-adherent cells. The adherent cells were replenished with RPMI media containing 20% FBS and incubated for 24 h. Non-adherent cells were further removed by washing with prewarmed RPMI medium. To induce M0 differentiation, the remaining adherent cells were treated with 50 ng/ml recombinant SCF, and 50 ng/ml GM-CSF (for M1 polarization) or 50 ng/ml M-CSF (for M2 polarization) (Cat. No. 573904, Cat. No. 572903, and Cat. No. 574804; BioLegend) for 7 d with 50% medium replenishment every 3 d. Upon differentiation, M0 cells were starved for 2 h as described above and given M1 or M2 polarization treatment as described above for 3 d.

Reprogramming of M2-TAMs to M1-TAMs

THP-1 and PBMC-derived M2-TAMs were treated with 100 μM SEP (CAS No. 17094-01-8; Career Henan Chemical Co.) every day for 3 d for reprogramming to M1-TAMs. M2-TAMs were also treated with DMSO (vehicle) and 5 ng/ml LPS and 20 ng/ml IFNγ as negative and positive controls, respectively.

PBMC-derived T-cell culture

PBMCs were incubated with 100 μg/ml DNase I solution (Cat. No. 7900; STEMCELL Technologies) at RT for 15 min and filtered through a 37-μm cell strainer. The filtered single-cell suspension was centrifuged at 800g for 5 min, and the collected pellet was resuspended at a density of 5 × 10⁷ cells/ml in the suspension medium composed of 2% FBS and 1 mM EDTA in PBS. T cells were then isolated using EasySep Human T Cell Isolation Kit and EasySep Magnet (Cat. No. 17951 and Cat. No. 18000; STEMCELL Technologies) following the manufacturer’s protocol. Isolated T cells were seeded at a density of 1 × 10⁶ cells/ml in RPMI medium supplemented with 10 ng/ml human recombinant IL2 (Cat. No. 78145.2; STEMCELL Technologies), 1 µg/ml anti-human CD3 antibody (Clone OKT3, Cat. No. 317326; BioLegend), and 1 µg/ml anti-human CD28 antibody (Clone CD28.2, Cat. No. 302934; BioLegend). The cells were maintained in a 37°C, 5% CO₂ humidified incubator for up to three subcultures with medium change every 3 d.

SEM

THP-1–derived TAM subsets were plated at a density of 1 × 10⁶ cells/ml on cellQART 12-well cell culture inserts with 0.4 μm PET membrane (Cat. No. 9310412; STERLITECH) and treated with SEP and relevant controls. Upon treatment, the membranes were washed with 1X PBS and fixed with 4% PFA. Then, the membranes were washed with deionized (DI) water and dehydrated using increasing concentrations of ethanol ranging from 25% to 100%. Afterward, the membranes were washed with 50% hexamethyldisilazane: ethanol solution followed by 100% hexamethyldisilazane. Later, the membranes were removed from the inserts, bound to the SEM sample stub with a carbon tape, and sputter-coated with gold for 15 s. The samples were then imaged using JEOL 7500F Scanning Electron Microscope.

Phalloidin staining

THP-1 cells were seeded at a density of 1 × 10⁶ cells/ml onto 12-mm coverslips (Cat. No. 1217N67; Thomas Scientific), precoated with poly-D-lysine (Cat. No. A003E; Thermo Fisher Scientific) and PureCol (Cat. No. 5005-100ML; Advanced BioMatrix), and singly placed into each well of 12-well plates. These cells were given SEP of control treatment and washed in 1X PBS and fixed with 3.7% formaldehyde in PBS for 10 min at RT. Cells were permeabilized with 0.1% Triton X-100 for 5 min and washed twice with PBS. After blocking in PBS containing 1% BSA for 30 min, cells were incubated for 20 min at RT with the phalloidin staining solution: Alexa Fluor 488 phalloidin (Cat. No. A12379; Thermo Fisher Scientific) and 1% BSA in PBS. Cells were washed with PBS and counterstained with the 4′,6-diamidino-2-phenylindole (DAPI) nuclear stain for 15 min at RT. Coverslips were then mounted on slides using Fluoromount-G Mounting Medium (Cat. No. 50-187-88; Thermo Fisher Scientific) and sealed with nail polish. Fluorescence images were captured using a Zeiss Axio Observer 7 microscope.

Immunofluorescence staining

THP-1 cells were plated on glass coverslips, polarized to TAMs, and given drug treatments as described above. Upon treatment, the coverslips were washed with 1X PBS, and cells were fixed in 4% PFA for 20 min at RT. The cells were permeabilized with 0.1% Triton X-100 for 15 min at RT and blocked with IF buffer (1.3 M NaCl, 70 mM Na2HPO4, 35 mM NaH2PO4, 77 mM NaN3, 1% BSA, 2% Triton X-100, and 5% Tween-20) containing 10% goat serum for 1 h. Cells were washed with PBS and incubated overnight at 4°C with primary antibody solution: antibody in IF buffer containing 3% saponin (Cat. No. 55-825-5100GM; Thermo Fisher Scientific). Antibodies used were as follows: TNFα (Cat. No. MA523720; Thermo Fisher Scientific), NOS2 (Cat. No. PA1-036; Thermo Fisher Scientific), CD163 (Cat. No. ab182422; Abcam), CD206 (Cat. No. NPB1-90020; Novus Biologicals), IL10 (Cat. No. MAB9184-100; R&D Systems), and Dectin-1 (Cat. No. PA583996; Thermo Fisher Scientific). Cells were then washed with PBS twice and incubated with secondary antibody solution (1:200 dil) for 2 h followed by DAPI stain for 15 min at RT. Coverslips were then mounted onto slides as described above. Fluorescence images were captured using an Olympus DP80 microscope and analyzed using ImageJ software.

Immunoblotting

Cell lysates were prepared using the following lysis buffer: 25 mM Tris–HCl (pH 8), 150 mM NaCl, 1 mM EDTA (pH 8), 1% NP-40, 5% glycerol, 1X PhosSTOP (Cat. No. 4906837001; Sigma-Aldrich), and 1X Protease Inhibitor Cocktail (Cat. No. 78425; Thermo Fisher Scientific). A total protein concentration of the cell lysates was quantified using Pierce BCA Protein Assay Kit (Cat. No. 23227; Thermo Fisher Scientific). Cell lysates were mixed with the sample buffer (with beta-mercaptanol) and boiled at 100°C for 10 min. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The separated proteins were then electroblotted to methanol-activated polyvinylidene fluoride (PVDF) membranes (Cat. No. IPVH00010; Sigma-Aldrich). Upon transfer, membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) and incubated overnight with the following primary antibodies: TLR2 (Cat. No. MA532787; Thermo Fisher Scientific), STAT1 (Cat. No. 9172T; Cell Signaling Technology), pSTAT1S727 (Cat. No. 8826S; Cell Signaling Technology), CD163 (Cat. No. ab182422; Abcam), CD206 (Cat. No. NPB1-90020; Novus Biologicals), STAT3 (Cat. No. 9139S; Cell Signaling Technology), pSTAT3Y705 (Cat. No. 9145S; Cell Signaling Technology), β-actin (Cat. No. A1978; Sigma-Aldrich), and GAPDH (Cat. No. 2118S; Cell Signaling Technology). Then, they were incubated with HRP-conjugated sheep anti-mouse IgG (Cat. No. NA931-1ML; GE Healthcare Life Sciences), donkey anti-rabbit IgG (Cat. No. NA934-1ML; GE Healthcare Life Sciences), or donkey anti-goat IgG (Cat. No. A16005; Thermo Fisher Scientific) secondary antibodies (1:5,000 dil). Next, the blots were developed with SuperSignal West Dura Extended Duration Substrate (Cat. No. 34076; Thermo Fisher Scientific) and imaged using the Syngene G:BOX F3 gel doc system.

Flow cytometry (FACS) analysis of cell surface markers

Cells were dissociated from the plates through incubation with PBS containing 5 mM EDTA for 15 min at 37°C, followed by gentle scraping. Cells were collected into 96-well V-bottom plates (Cat. No. 5665-1101; USA Scientific) and centrifuged at 1,000 rpm for 5 min. Cell pellets were washed with FACS buffer (PBS, 2% FBS) and blocked for 30 min on ice in the blocking buffer: 2% FBS, 2% goat serum, 2% rabbit serum, and 10 μg/ml human immunoglobulin G (IgG). Cells were then incubated with fluorochrome-labeled antibodies prepared in FACS buffer for 1 h on ice. The antibodies used are as follows: CD68 (Cat. No. 333821; BioLegend), CD40 (Cat. No. 334305; BioLegend), CD80 (Cat. No. 305205; BioLegend), CD163 (Cat. No. 333609; BioLegend), CD206 (Cat. No. 321109; BioLegend), anti-mouse F4/80 (Cat. No. 123118; BioLegend), anti-mouse CD80 (Cat. No. 104706; BioLegend), anti-mouse CD163 (Cat. No. 155306; BioLegend), anti-mouse NK1.1 (Cat. No. 61-5941-80; Thermo Fisher Scientific), and anti-mouse NKp46 (Cat. No. FAB2225F-025; R&D Systems). Cells were washed twice with FACS buffer and resuspended in Dulbecco’s phosphate-buffered saline containing 2% formaldehyde. Samples were assayed on the BD FACSCanto II system, followed by analyses with FlowJo software.

FACS analysis of intracellular markers

Cells were treated with 1 μg/ml Brefeldin A (Cat. No. B7450; Thermo Fisher Scientific) for 5 h at 37°C. Upon treatment, cells were collected into 96-well V-bottom plates and centrifuged as described above. The samples were then incubated with 1X fixation/permeabilization buffer (Cat. No. FC007; R&D Systems) for 12 min at 4°C. After fixation, samples were centrifuged at 1,600 rpm for 5 min and washed with the following permeabilization/washing buffer: PBS, 2% FBS, and 0.1% Triton X-100. Samples were then blocked with blocking buffer containing 0.1% Triton X-100 for 10 min. After blocking, samples were incubated with fluorochrome-labeled antibodies prepared in permeabilization/washing buffer for 45 min at 4°C. The following antibodies were used: anti-mouse IFNγ (Cat. No. IC485F-025; R&D Systems), anti-mouse IL12 (Cat. No. 505206; BioLegend), anti-mouse IL10 (Cat. No. 505006; BioLegend), and anti-mouse perforin (Cat. No. 11-9392-80; Thermo Fisher Scientific). After antibody incubation, samples were washed with permeabilization/washing buffer, resuspended in Dulbecco’s phosphate-buffered saline containing 2% formaldehyde, and assayed on the BD FACSCanto II system, followed by analyses with FlowJo software.

Measurement of BH₄ production

THP-1–derived TAMs plated in six wells were used for the measurement of BH4. Cells were washed with ice-cold PBS to remove remaining media. Upon washing, cells were scraped gently, and the cell pellets were collected into Eppendorf tubes. The cell pellets were vortexed for 10 s and flash-frozen in liquid nitrogen (N2). Then, the pellets were thawed at RT. The process was repeated five times. Then, the pellets were centrifuged at 1,000 rpm for 5 min, and the supernatants were collected into fresh tubes. The BH4 level in cell lysate was measured using an ELISA kit (Cat. No. abx354211; Abbexa) following the manufacturer’s protocol. Cellular BH4 levels were normalized using the total protein concentration in cell lysates.

Live-cell imaging of NO and PA

Intracellular NO and PA levels were detected in live cells with the help of cell-permeable fluorescent dyes. Cells grown in phenol red and serum-free media were stained with 20 μM diaminorhodamine-4M acetoxymethyl ester (DAR-4M AM) (Cat. No. ALX-620-069-M001; Enzo) or 20 μM PolyamineRED (Cat. No. FDV-0020; DiagnoCine) according to the manufacturers’ protocol to detect NO and PA, respectively. Fluorescence images were captured using an Olympus DP80 microscope and analyzed using ImageJ software.

Measurement of NO levels

Nitric Oxide Fluorometric Assay Kit (Cat. No. K252; BioVision) was used to quantify the NO levels present in the CM. The samples were filtered through a 10-kD cutoff Microcon filter (Millipore) to remove proteins present in the CM. The flow-through was reacted with the assay reagents in the dark according to the manufacturer’s protocol, and the fluorescence intensity was measured at the wavelengths of Ex/Em = 360/450 nm.

Measurement of PA levels

PA levels in the CM were quantified using Fluorometric Total Polyamine Assay Kit (Cat. No. K475; BioVision) according to the manufacturer’s protocol with modifications. This kit determines the level of hydrogen peroxide produced through oxidation of polyamines by spermine/spermidine oxidase in the kit. To remove high background levels of hydrogen peroxide produced by macrophages before the assay, the CM were pretreated with catalase (Cat. No. C40-100MG; Sigma-Aldrich) at 100 μg/ml and incubated at 37°C for 1.5 h. Proteins were precipitated with Sample Clean-up Solution provided by the kit and removed by filtration through a 10-kD cutoff Microcon filter. The flow-through was reacted with the assay reagents in the dark according to the manufacturer’s protocol, and the fluorescence intensity was measured at the wavelengths of Ex/Em = 535/587 nm.

Measurement of cytokine secretion

Cells were cultured in RPMI serum-free media for 2 d after treatment, and the CM were collected. Secreted cytokines in the CM were quantified using ELISA kits according to the manufacturers’ protocol. The ELISA kits used were as follows: IL12 (Cat. No. D1200; R&D Systems), IL6 (Cat. No. D6050; R&D System), IL1β (Cat. No. DLB50; R&D System), TNFα (Cat. No. DTA00D; R&D System), IL10 (Cat. No. D1000B; R&D Systems), and TGFβ (Cat. No. ab108912; Abcam).

Antigen presentation assay

THP-1–derived TAMs were detached from the plates, collected into 96-well V-bottom plates, and centrifuged as described before. The cells were then pulsed with 20 μg/ml OVA323–339 peptide (Cat. No. RP10610-1; GenScript) for 2 h at 37°C and 5% CO2. Cells were then washed twice with FACS buffer (see above) to remove residual OVA peptides and incubated with blocking buffer for 30 min at 4°C. Samples were stained with fluorescein isothiocyanate (FITC)–labeled HLA-DR antibody (Cat. No. 307604; BioLegend) in FACS buffer for 1 h at 4°C and analyzed by flow cytometry.

TAM, T-cell, and cancer cell co-culture model

A triple co-culture model of breast cancer cells, TAMs, and T cells was developed to emulate and study the tumor–immune interactions within the TME.

Transwell co-culture system

THP-1 cells were seeded on cellQART 12-well inserts (0.4 μm pore size and PET membrane) at a density of 3 × 10⁵ cells/ml and subjected to differentiation, polarization, and reprogramming as described above. After establishment of macrophages, the inserts were washed with fresh RPMI media, and breast cancer cells (CA1d, SKBR3, or BT-474) in RPMI media were seeded on top of THP-1 cells at a ratio of 1:2 (cancer cells: THP-1 cells). Into separate bottom wells of 12-well plates, the equal amounts of breast cancer cells were seeded in RPMI media. Once cells were attached, PBMC-derived T cells were seeded on top of cancer cells at a ratio of 1:2 (cancer cell: T cell). The inserts containing cancer cells and THP-1 were placed on top of the wells containing cancer cells and T cells. The co-cultures were incubated in a humidified incubator set at 37°C with 5% CO2 for further experiments.

Direct co-culture system

Monocytes and T cells isolated from the same PBMCs were used to establish the direct co-culture system. Monocytes were seeded on 12-well plates at a density of 1.5 × 10⁶ cells/ml, differentiated, polarized into relevant TAM subsets, and reprogrammed as described above. The media were removed, and cells were washed with fresh RPMI serum-free media. Breast cancer cells and T cells were sequentially added to the 12-well plates at a ratio of 1:2:2 (cancer cell: TAM: T cell) in the same RPMI media. The co-culture was incubated in a humidified incubator set at 37°C with 5% CO₂ for further experiments.

T-cell proliferation assay

PBMC-derived T cells were washed with PBS and resuspended at a density of 1 × 10⁶ cells/ml. The cells were then stained with the CellTrace CFSE dye (Cat. No. C34554; Thermo Fisher Scientific) following the manufacturer’s protocol. CFSE-labeled T cells were directly co-cultured with PBMC-derived TAMs and cancer cells as described above and incubated for 4 d. T cells were isolated and stained with fluorochrome-labeled anti-human CD3 (Cat. No. 317342; BioLegend) and anti-human CD8a (Cat. No. 301014; BioLegend) antibodies as described above. The cytotoxic T-cell proliferation, indicated by changes in CFSE signals, was measured by flow cytometry.

Measurement of IFNγ production

The triple co-culture, composed of cancer cells, TAMs, and T cells, was established as described above and incubated for 2 d. After incubation, the cells were treated with 1 μg/ml Brefeldin A for 4 h to block protein secretion. T cells were isolated and stained with anti-human CD3 and CD8a antibodies (surface staining) followed by intracellular staining with anti-human IFNγ (Cat. No. IC285F-100; R&D Systems) antibody, as described before, and analyzed by flow cytometry.

Degranulation assay

CD107a marker expression was measured to analyze the degranulation of cytotoxic T cells. The triple co-culture system was set up as previously described and incubated for 2 d. Upon incubation, 10 μl of anti-human CD107a antibody (Cat. No. 328606; BioLegend) was added to each well and incubated in a humidified incubator set at 37°C with 5% CO₂. After 1 h of incubation, 1X eBioscience Monensin Solution (Cat. No. 00-4505-51; Thermo Fisher Scientific) was added to each well and the incubation was continued for three more hours to block protein transport. T cells were then isolated and stained with CD3 and CD8a antibodies, as previously described. CD107a expression in cytotoxic T cells was measured by flow cytometry.

Measurement of extracellular ATP

During ICD—a form of apoptosis—dying cells release ATP to induce immunogenic phagocytosis. Thus, the increase in secreted ATP is used to determine the occurrence of ICD. A luminescent ATP detection assay kit (Cat. No. ab113849; Abcam) was used to quantify ATP released from cells. The co-culture system was set up as described above, and 100 μl of CM from the bottom well (seeded with cancer cells and T cells) was analyzed for ATP levels, in comparison with CM of monocultured TAMs, T cells, and cancer cells as controls. ATP concentration was quantified using the standard following the manufacturer’s protocol.

Cell cycle analysis

Cancer cells were labeled with CellTracker Green CMFDA Dye (Cat. No. C7025; Thermo Fisher Scientific) according to the manufacturer’s protocol. The labeled cancer cells were directly co-cultured with PBMC-derived TAMs and T cells as described above and incubated for 4 d. Cells were harvested using 0.05% Trypsin (Cat. No. 25-300-062; Thermo Fisher Scientific), washed with PBS, and centrifuged at 1,000 rpm for 5 min. The pelleted cells were fixed with precooled 70% ethanol for 2 h at 4°C. Cells were centrifuged at 4,000 rpm for 2 min, resuspended in PBS containing 0.25% Triton X-100, and incubated on ice for 15 min. Cells were centrifuged and resuspended in PBS containing 10 μg/ml ribonuclease (RNase) A and 20 μg/ml propidium iodide (PI) (Cat. No. R4642-10MG and Cat. No. P4170-10MG; Sigma-Aldrich) for the staining of DNA. Cells were transferred to FACS tubes, incubated in the dark for 30 min at RT, and analyzed for cell cycle profiles by flow cytometry.

Apoptosis assay

The direct co-culture model was set up using CMFDA-labeled cancer cells as described before and incubated for 4 d. Adherent cells were harvested, washed with FACS buffer, and centrifuged at 1,000 rpm for 5 min. The pelleted cells were then resuspended at a density of 1 × 10⁶ cells/ml in Annexin V Binding Buffer: 10 mM Hepes, 150 mM NaCl, and 2.5 mM CaCl2 in PBS. Resuspended cells were transferred to FACS tubes, stained with 5 μl of Annexin V (Cat. No. A35110; Thermo Fisher Scientific) and 20 μg/ml of PI, and incubated in the dark for 15 min at RT. Each tube was replenished with 400 μl of Annexin V Binding Buffer and analyzed by flow cytometry.

Animal study

All in vivo experiments were performed in compliance with the Guide for the Care and Use of Laboratory Animals (National Research Council, National Academy Press, Washington, D.C., 2010) and with the approval of the Institutional Animal Care and Use Committee of the University of Toledo, Toledo, OH (Protocol No: 108658), and Case Western Reserve University, Cleveland, OH (Protocol No. 2022-0080).

For the tumor treatment study, 2-mo-old female MMTV-neu/FVB (unactivated) (n = 14) mice were obtained from the Jackson Laboratory (ID. IMSR_JAX:002376), housed under regular conditions, and given ad libitum access to acidified water and regular chow. The mice were maintained until spontaneous mammary tumors became palpable (∼5 mm long, 6–14 mo). Upon tumor onset, mice were divided into vehicle (DMSO) versus SEP (10 mg/kg) treatment groups (70, 71). The drugs were dissolved in acidified drinking water and administered to mice ad libitum for 6 wk (75). The tumor growth, body weight, and the morbidity of the animals were monitored twice a week. Tumor volume was calculated by the modified ellipsoidal formula: Volume = (Length x Width x Width) x ½. At the end of treatment, mice were euthanized, and mammary tumors were processed for further analyses as described below. As a complementary model, 6-wk-old FVB/NJ mice (n = 20) were obtained from the Jackson Laboratory and treated with DMSO or SEP (10 mg/kg) through drinking water until they became 5 mo old. These animals were injected with NT2.5 cells derived from mammary tumors of MMTV-neu/FVB (57) at around 2.5 × 10⁶ cells/mouse through nipples and treated with DMSO or SEP for another 100 d. The tumor growth, body weight, and the morbidity of the animals were monitored three times a week. At the end of treatment, mice were euthanized, and mammary tumors were processed for further analyses.

For the tumor prevention study, 4-wk-old female MMTV-neu/FVB (unactivated) (n = 20) mice were divided into the vehicle (DMSO) versus SEP (1 mg/kg) treatment group. The drugs were dissolved in acidified drinking water and administered to mice ad libitum for 7 mo. Tumor incidence was monitored, and the percentage of tumor-free mice was quantified. The body weight, tumor incidence, latency, and size were monitored twice a week, and urine and fecal samples were collected once a week. At the end of treatment, mammary tumors, spleens, livers, and blood were harvested and processed for further analyses.

Profiling of macrophage cells in tumors and spleens

Freshly harvested tumors and spleens were processed for the profiling of resident macrophages. Tumors and spleens were weighed and reacted in the digestion mixture (10 ml/g of tissue, 3 mg/ml collagenase A [Cat. No. 10103578001; Sigma-Aldrich], and 25 μg/ml DNase I [Cat. No. 10104159001; Sigma-Aldrich]) in HBSS (Cat. No. 14025092; Thermo Fisher Scientific) with gentle motions on a platform shaker for 45 min at 37°C (76). To stop the enzymatic digestion, the samples were treated with 10 ml of staining buffer (1% [wt/vol] BSA in PBS). The cell suspension was then filtered through a 100-μm cell strainer, and the volume was adjusted to 20 ml with staining buffer. Then, cells were pelleted by centrifugation at 500g for 7 min at 4°C. To remove red blood cells, the pelleted cells were suspended in 3 ml of 1X RBC lysis buffer (Cat. No. 00-4300-54; Thermo Fisher Scientific) and incubated on ice for 10 min. A volume of 30 ml of staining buffer was added, and cells were centrifuged and resuspended in 1 ml of staining buffer. To block Fc receptors (to avoid unwanted antibody binding), cells were treated with 2 μl of anti-mouse CD16 (FcγII)/CD32 (FcγIII) antibody (Cat. No. 14-0161-82; Thermo Fisher Scientific) and incubated on ice for 30 min with mixing at 10-min intervals. A volume of 4 ml of staining buffer was added, and samples were centrifuged at 500g for 5 min at 4°C. Then, cell pellets were resuspended in 1 ml of staining buffer (77). Cells were then stained with relevant fluorochrome-labeled antibodies, as previously described, and analyzed by flow cytometry.

PBMC isolation from mouse blood

At the end of the tumor prevention experiment, MMTV-neu/FVB mice were grouped into four groups (DMSO with tumors, DMSO without tumors, SEP with tumors, and SEP without tumors). Blood was collected from submandibular veins, pooled by group, and mixed with the equal volume of PBS-EDTA solution (Cat. No. J60893.K3; Thermo Fisher Scientific). To isolate mononuclear cells, the diluted blood was added on top of Lymphoprep medium (Cat. No. 07851; STEMCELL Technologies) within a SepMate-15 centrifuge tube (Cat. No. 85415; STEMCELL Technologies) and spun at 1,200g for 10 min at room temperature for density gradient centrifugation. Mononuclear cells accumulated at the interface between the top serum and bottom Lymphoprep layers were carefully collected and transferred into a separate centrifuge tube. Cells were washed in PBS plus 2% FBS twice, resuspended in RPMI-1640 medium, and cryopreserved until single-cell sequencing.

Single-cell sequencing

Single-cell sequencing was performed with GEM-X, Chromium Single Cell Gene Expression (3′ GEX V3.1) chips on 10X Genomics Chromium X Processor at the Discovery Lab in the Global Center for Immunotherapy and Precision Immuno-Oncology at Lerner Research Institute, Cleveland Clinic. Fastq files were mapped to the GRCh38 reference human genome using Cellranger (v5.0.0) (78). Cells containing less than 600 genes and/or more than 30% mitochondrial and ribosomal genes were removed. Sample-specific Seurat objects were created using Seurat (v4.3.0) (79), then normalized using Seurat’s SCTransform method. Samples were integrated based on variable features using Seurat’s IntegrateData function. To help predict cell types, the Seurat object was uploaded to BioTuring BBrowserX (80 Preprint), and cells were annotated using their deep learning–based mouse cell–type prediction model (subcell type [version.2] model).

Immunohistochemistry

To determine the expression of specific markers in tissues harvested from mice, paraffin-embedded sections were analyzed by immunohistochemistry. Briefly, sections were deparaffinized, hydrated, and treated with antigen unmasking solution (Cat. No. H-3300-250, pH 6.5; Vector Lab., Inc.) or with Tris–EDTA buffer (10 mM Tris base, 1 mM EDTA solution, and 0.05% Tween-20, pH 9.0), which had been heated to 95–100°C in a pressure cooker. After being blocked with non-immune goat serum, sections were processed for immunofluorescence staining as described below.

Immunofluorescence staining and imaging

Immunofluorescence staining/imaging was performed as described previously (21, 81). Samples were incubated with a primary antibody for overnight at 4°C in a humidified chamber. Primary antibodies used for immunofluorescence staining of mammary tumors were antibodies against mouse CD80 (Cat. No. 104705; BioLegend), CD163 (Cat. No: ab182422; Abcam), CD8 (Cat. No. PA579011; Thermo Fisher Scientific), CD4 (Cat. No. 13-9766-82; Thermo Fisher Scientific), and FOXOP3 (Cat. No. MAB8214; Bio-Techne). After intensive washing (three times, 15 min each) in 0.1% BSA, 0.2% Triton X-100, 0.05% Tween-20, and 0.05% NaN3 in PBS, fluorescence-conjugated secondary antibodies (Molecular Probes) were added for 2 h at room temperature. Nuclei were stained with 0.5 ng/ml DAPI. After being mounted with anti-fade solution, epifluorescence imaging was performed on the Leica Thunder Imaging platform with Leica LAS X Life Science Microscope software.

Image analysis

Quantification of fluorescence signal in micrographs captured with 10x objective was performed with ImageJ software (NIH) referring to the owner’s manual (http://imagej.net/docs/guide/146.html) as described previously (21). For particle analysis to count positively stained immune cells, images were converted to gray-scale images, and the same threshold ranges were set for each marker analysis. Using the Analyze Particles function, the numbers of positive cells were automatically counted. For each tumor section, 8–10 regions were analyzed. The mean value was represented as the number of positive cells per field. The statistical significance of the data was further evaluated using GraphPad Prism version 10 software (see the statistics section).

Statistical data analysis

The experimental results are presented as the mean ± SEM. All the experiments were performed in replicates (n ≥ 3), and unless otherwise indicated, two-tailed t tests were performed to obtain the statistical significance of the mean difference. P-values ≤ 0.05 were considered statistically significant. Flow cytometry data analyses were performed using FlowJo version 10.5. Imaging analyses were performed on ImageJ software. All statistical analyses were carried out using GraphPad Prism version 10.