Mice
Truncated il2rg alleles of male B6.129S4-Il2rgtm1Wjl/J (IL2Rγnull) mice were backcrossed into female NOD.CB17-Prkdcscid/JNarl (NOD.SCID) mice for ten generations to obtain NOD.Cg-PrkdcscidIl2rgtm1Wjl/ YckNarl (ASID) mice at Dr. Chun-Keung Yu’s Laboratory (National Cheng Kung University, Taiwan). ASID mice were used at 4-16 weeks of age for the experiments. BALB/cByJNarl (BALB/c) mice, CAnN.Cg-Foxn1nu/CrlNarl (Nude) mice and NOD/ShiLtJNarl (NOD) mice were purchased from the National Laboratory Animal Center (NLAC), Narlabs, Taiwan. All animal procedures were reviewed and verified by the Institutional Animal Care and Use Committee NLAC (IACUC; IACUC numbers NLAC-109-M-013). CO2 Euthanasia were performed at experimental endpoints in accordance with the NLAC SOP based on AVMA guideline 2020. Post approval monitoring (PAM) programs were implemented during the research. All reported methods are in accordance with ARRIVE guidelines. All methods were performed in accordance with relevant guidelines and regulations.
Concanavalin A (Con A)-induced lymphocyte proliferation test
The Con A-induced lymphocyte proliferation of spleen cells of BALB/c and ASID mice were assessed by 5-bromo-2’-deoxyuridine (BrdU) labeling and FACS analyzing. Spleens harvested from BALB/c and ASID mice were squeezed with a 70-mm cell strainer (BD Falcon) and the red blood cells (RBCs) in filtered cells were lysed subsequently with RBC lysis buffer (BD Pharm Lyse™ lysing solution, BD Bioscience). Isolated spleen cells (1´106 cells/mL, 200 mL/well) were suspended in RPMI1640 medium (GIBCO) with 10% FBS (Hyclone) and then activated with indicated concentrations of Con A (C5275, Sigma-Aldrich). After 36 hours of activation, 20 mL of BrdU-containing RPMI 1640 medium (BrdU: 200 mM) were added for a further 12 hours of incubation. The labelled spleen cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 15 min at room temperature and treated with permeabilization buffer (0.1% Triton X-100 in PBS) for 20 min at room temperature. The resulting cells were stained with PE-anti-mouse CD3 (BD Bioscience) and FITC-anti-BrdU mAb and then analyzed by FACS (FACSCalibur, BD).
Induction of diphtheria-specific immunoglobulin
BALB/c and ASID mice were injected with 30 ml/mouse PEDIACEL® vaccine (Sanofi Pasteur Limited), which is specific to diphtheria, tetanus, whooping cough (pertussis), polio and Hib disease (Haemophilus influenzae type b) or PBS. Twenty-seven days after vaccination, the sera were collected and subjected to diphtheria-specific immunoglobulin quantification with a diphtheria ELISA kit (IBL, German) following the manufacture’s protocols.
Generation of immune humanized mice
Human CD34+ cord blood cells (hHSC, Cat. 2C-101) and human peripheral blood mononuclear cells (hPBMC, Cat. CC-2702) were purchased from LONZA (Basel Switzerland). At indicated ages, female ASID mice were conditioned with whole body irradiation (2 Gy) prior receiving hHSCs and hPBMCs. Percentages of hCD45+ cells in peripheral blood leukocytes (PBLs) from mice received hHSCs were analyzed 90 days (D90) post transfer (PT). The hHSCs-transferred host mice with more than 25% hCD45+ cells in PBLs were defined as hHSC-HIS immune humanized mice. Percentages of hCD45+ cells in PBLs from hPBMCs-transferred host mic were analyzed 7 days PT. Mice that received hPBMCs with hCD45+ cells in PBLs were considered successful hPBMC-HIS mice.
Establishment of orthrotropic MDA-MB-231 model
The human triple negative breast cancer cell line, MDA-MB-231, was obtained from the American Type Culture Collection (ATCC). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), TrypLE™ Express Enzyme, and Dulbecco's phosphate-buffered saline (DPBS) were purchased from ThermoFisher (MA, USA). Matrigel matrix was purchased from BD Bioscience (MA, USA). The MDA-MB-231 cells were cultured in DMEM supplemented with 10% FBS and were maintained in a humidified incubator at 37°C containing 5% CO2. The MDA-MB-231 cells were tested for absence of Mycoplasma spp. before inoculation into humanized and naïve ASID mice. Cells were harvested and resuspended in a 1:1 mixture of DPBS and Matrigel. Three million cells were injected subcutaneously into the right lower flank (orthotopic: the 4th mammary gland fat pad) of each mouse with a 29-gauge needle. After cell inoculation, the length (L) and width (W) of tumors were measured twice per week and the tumor volumes (V) were estimated as V (mm3) = (L x W2)/2.
Antibodies
Antigen
|
Conjugation
|
Cat. No
|
Manufactures
|
Mouse CD3
|
PE
|
553240
|
BD Pharmingen™
|
Mouse IgD
|
APC
|
560868
|
BD Pharmingen™
|
Mouse CD49b
|
FITC
|
561067
|
BD Pharmingen™
|
Mouse CD11c
|
FITC
|
561045
|
BD Pharmingen™
|
Mouse CD11b
|
BV605
|
563015
|
BD Horizon™
|
Mouse Ly6G/C
|
PE-Cy7
|
565033
|
BD Pharmingen™
|
Human CD45
|
APC-Cy7
|
557833
|
BD Pharmingen™
|
Human CD3
|
A488
|
53-0037-42
|
Bioscience™
|
Human CD56
|
APC
|
555518
|
BD Pharmingen™
|
Human CD19
|
ef506
|
69-0193-82
|
eBioscience™
|
Human CD14
|
A647
|
562690
|
BD Pharmingen™
|
Human CD45
|
Cy5
|
19-0459-42
|
eBioscience™
|
Human CD3
|
A647
|
557706
|
BD Pharmingen™
|
Human CD4
|
A488
|
557695
|
BD Pharmingen™
|
Human CD8
|
PE
|
555635
|
BD Pharmingen™
|
Human Pan Cytokeratin
|
A488
|
53-9003-82
|
eBioscience™
|
Human CD3
|
PE
|
561803
|
BD Pharmingen™
|
Human CD33
|
A647
|
366625
|
Biolegend
|
Human CD14
|
PE
|
562691
|
BD Pharmingen™
|
BrdU
|
FITC
|
11-5071-42
|
eBioscience™
|
Flow cytometer analysis
PBLs from host mice at indicated time points described in the figure legends were collected from facial veins (Submandibular) using Golden Lancets (4 mm, Medipoint Inc.). Spleens and MDA-MB-231 tumors from host mice at endpoint were collected. The spleens were gently torn apart with forceps in HBSS. The suspended splenocytes were treated with ACK RBC-lysis buffer and distributed in HBSS+5% FBS. The TILs were prepared with a Tumor Dissociation Kit (Cat. 130-095-929, Miltenyi Biotec) according to the manufacturer’s instructions. All resulting cells were stained with fluorochrome-conjugated antibodies, which are described in the figure legend shown in the antibody list. The antibody-stained sample cells were subjected to flow cytometer analysis with an LSR Fortessa (BD).
Biomarker determinations
Serum/plasma from mice were collected as described previously/in the figure legends. The samples were subjected to biomarker determination with the human cytokine/chemokine magnetic bead panel (Cat. HCYTMAG-60K-PX41, EMD Millipore) by the immunological analysis service of the National Laboratory Animal Center, Taiwan. The determination procedures were performed according to the manufacture instructions, and the data were acquired with a Luminex® 200™ System with xPONENT® Software (Luminex). The readouts were analyzed by MILLIPLEX™ Analyst using 5-parameter logistic standard curve fitting models as described in the manufacture’s manual.
Pathological examination
Tissues (livers, lungs, kidney, pancreases, hearts, and gastrointestinal tracts) and tumors from host mice were collected for pathological observations. The histological examination for H&E staining was performed as described previously (28). Inflammations of livers and lungs were evaluated. The severity of inflammation in livers was evaluated by Salomao’s method (29). The liver inflammation was scored as biliary cell apoptosis, bile duct damage, cholestasis (0, none; 1, mild; 2, moderate; 3, severe), portal inflammation with or without interface hepatitis (0, none; 1, mild; 2, moderate; 3, severe), lobular inflammation and hepatocyte apoptosis (0, none; 1, mild; 2, moderate; 3, severe), venulitis (0, none; 1, mild; 2, moderate; 3, severe), ductopenia, fibrosis and ductular proliferation (0, none; 1, mild; 2, moderate; 3, severe), and other concurrent findings (0, none; 1, mild; 2, moderate; 3, severe). The maximum score was 18. The severity of inflammation in tumor and lung tissues were scored as degree of inflammation (0, none; 1, mild; 2, moderate; 3, severe) and distribution of inflammatory area (0, none; 1, single or few; 2, multifocal; 3, diffuse). The maximum score was 6.
Immunofluorescent staining
Tissues for immunofluorescent staining were prepared as described previously (30). Indicated fluorescent dye-conjugated antibodies are described in the figure legends. Three sections of each tissue sample from each host mouse were prepared.
Statistical analysis
The primary outcomes of this study were the frequency of leukocytes in PBL, spleen, and TME of MDA-MB-231 engrafted immune humanized mice. The second outcomes were the tumor volumes of tumor-bearing mice, expression of immune cells in humanized mice and relative control mice, as well as percentage of BrdU+ splenocytes and diphtheria-specific antibodies in ASID and BALB/c mice. Differences in variables between two groups were analyzed by Student t-test or Mann-Whitney U test after normality test. Data presented as box and whisker plots show the minimum score, first quartile, median, third quartile, and maximum score. The difference in the survival rate between hHSC-ASID and hPBMC-ASID mice was analyzed by log-rank test. Data presented as bar charts show the mean and standard deviation (SD). P values < 0.05 were considered significant, and the P values of each result are shown in the figures. GraphPad Prism software (ver. 8.4.0; La Jolla, CA, USA) was used for the statistical analysis.