Dasatinib

Class II tetramer enrichment strategy was performed per Moon and colleagues [70 (link)]. Briefly, cells from all the peripheral lymphoid organs (spleens and all the lymph nodes) were harvested from 6–10-week-old naïve DO-WT or DO-KO and DR1⁺ DO-WT or DR1⁺ DO-KO mice. Cells were stained with PE-conjugated MOG tetramers or PE-conjugated CII tetramers for 2 hours at 37°C in RPMI + 2% FBS + 0.1% azide in the presence of 50 nM Dasatinib (Cell Signaling Technology, Danvers, MA). Tetramer positive cells were isolated via anti-PE bead positive selection (Stemcell Technologies, PE positive isolation kit) and subjected to flow cytometry analysis.

Antibodies directed against EphA2 (WB analysis for total EphA2), Chlamydia Hsp60, GP96, PARP H-250, Pan Cadherin, GAPDH were purchased from Santa Cruz biotechnology. Antibodies against N-terminal EphA2 (D4A2) for WB and IF studies, pEphA2 (phospho EphA2 Ser897), pERK, pAkt, pPI3K, p85-PI3K, total Akt and total ERK were from Cell Signaling technology. Antibodies against N-terminal EphA2 (FACS, WB and IF), EphB4, PDGFRβ and IgG (control) were from R&D systems. Antibody against PDI was bought from Thermo Scientific. Anti-β-Actin and anti-Flag antibodies were purchased from Sigma-Aldrich. Phalloidin was bought from Invitrogen. Draq5 was from BioStatus Limited, UK. Polyclonal serum against IncA (anti-rabbit) was obtained through Gramsch laboratories. Inhibitors for PI3 kinase (LY294002), MEK1/2 (UO126) and EphA2 (dasatinib) were bought from Cell Signaling. Recombinant human EphA2 (rhEphA2) and recombinant human Ephrin-A1 homodimer (rhEphrin-A1 fused with IgG1-Fc) were purchased from R&D systems. Recombinant human prohibitin-His (rhPHB) also possess a His tag and is a control for rhEphA2. As a control for rhEphrin-A1, recombinant IgG1-Fc was bought from Life Technologies.

B-ALL cells (2.5 × 10⁵) were injected via the tail vein into NOD/SCID/Gamma-immunodeficient mice (Duke Cancer Center, Durham, NC, USA). Two days after transfer, mice were treated with vehicle (corn oil; Sigma-Aldrich) or tamoxifen (120 mg/kg per day; Sigma-Aldrich) by i.p. injection for four consecutive days. GFP+B-ALL cells were observed in spleen and bone marrow by flow cytometry. In some cases, animals were treated with 2-DG (500 mg/kg per day; Sigma-Aldrich) or Dasatinib (10 mg/kg per day; Cell Signaling Technologies) through oral gavage, as indicated.

At passage 2, EnSC and gland-like organoid pellets were mixed at a ratio of 1:1 (v/v) and ice-cold PureCol EZ Gel (Sigma-Aldrich) added at a ratio of 1:20 (cell pellet: hydrogel). Samples were kept on ice until plating. The suspension was aliquoted in 20 µl volumes using ice-cold pipette tips into a 48-well plate, one droplet per well, and allowed to cure in the cell culture incubator for 45 min. Expansion medium supplemented with 10 nM E2 was overlaid and the medium was refreshed every 48 hr. For decidualization experiments, assembloid cultures were grown in expansion medium supplemented with E2 for 4 days to allow for growth and expansion. Assembloids were then either harvested or decidualized using different media as tabulated in Supplementary file 1: Table 1 for a further 4 days. Again, the medium was refreshed every 48 hr and spent medium stored for further analysis. For tyrosine kinase inhibition, MDM was supplemented with 250 nM dasatinib (Cell Signaling Technology, Leiden, NL).

QFL T cells were enriched using a QFL tetramer (FL9-Qa-1b) synthesized by the NIH tetramer core facility. Spleen cells were isolated as stated above and incubated with 50 nM of Dasatinib (Cell signaling #9052) at 37°C for 30 minutes. Cells were then washed and incubated with PE-QFL tetramer (1:200) for 1 hour at RT. Cells were washed, resuspended in 150ul FACS buffer with 100 ul of anti-PE microbeads (Miltenyi Biotec), and incubated for 20 minutes at 4°C. Cells were then washed and passed through an LS magnetic column (Miltenyi Biotec). Enriched cells were stained with antibodies for B220, CD8α, CD8β, and Vα3.2. Tetramer+ QFL T cells were gated as B220⁻ CD8α⁺QFL Tet⁺Vα3.2⁺. CountBright beads (Invitrogen) were used in each sample to measure total cell numbers in the enriched and unenriched samples.

The ROCK inhibitor Y27632 was obtained from Enzo (ALX-270-333) and the Myosin II inhibitor Blebbistatin from Cambridge Bioscience (CAY13165). PP2 and IWP2 were from Tocris (#3533, #1407) and Dasatinib was from Cell Signalling Technologies (#9052). Y27632 was dissolved in sterile Milli-Q water; all other inhibitors were dissolved in sterile DMSO.