Lentiviral Marking of Patient Derived Acute Lymphoblastic Leukaemic Cells Allows in Vivo

Lentiviral marking of patient derived acute lymphoblastic leukaemic cells allows in vivo tracking of disease progression

Supplementary Data

Supplementary methods

Patient derived material

The patient derived material was collected as part of the initial diagnostic investigation of patients. It was collected, stored and used with written informed consent according to approvals given by the local institutional review boards and the Declaration of Helsinki. Samples were retrieved from Newcastle HaematologicalBioBank under the generic BioBank approval given by the Newcastle & North Tyneside Ethics Committee (REC reference number: 07/H0906/109).

Home Office approval for animal research

Animal studies were performed by researchers who had all completed approved Home Office training and held current Personal Licences under the Animals (Scientific Procedures) Act 1986. All work was conducted in accordance with the Home Office Project Licence PPL60/3846.

Washing procedure

On Day 1 following transduction, 350 μl supernatant was removed from the transduced cells. Cells were resuspended in 850μl medium followed by the addition of a further 20 ml medium (RPMI, 10% fetal calf serum). Cells were centrifuged 300g for 10 minutes. The supernatant was aspirated using a plastic pipette (to avoid the risk of sharps related injuries) and the cells resuspended to repeat the above wash procedure. Following the second wash, cells were resuspended in 1 ml SFEM supplemented as for the transduction experiment. The wash procedure was repeated on Day 2.

Immunohistochemical staining of organs from mouse engrafted with L578-SLIEW

Organs harvested from the primary recipient mouse of transduced L578 blasts were fixed in formalin, paraffin embedded and sectioned at 4 µm. Serial sections were stained for CD10, CD79a and TdT in the Department of Pathology, Royal Victoria Infirmary, Newcastle using either a Ventana Benchmark XT or Benchmark Ultra and VentanaUltraview detection system.

EGFP expressing cells were visualised directly by fluorescence microscopy following mounting with DAPI counterstain, x400 magnification.

Supplementary results

Table S1.Details of transductions

Summary of transduction experiments performed during the development of the protocol. PB – primary peripheral blood, BM1 – primary bone marrow, BM2 – primografted bone marrow,αMEM – alpha modified Minimal Essential Medium, SFEM – Serum free expansion medium (Stem Cell Technologies), SCF – stem cell factor, IL-7 – Interleukin -7, HS-5 CM – HS-5 conditioned medium, HS – horse serum, Il-3 – Interleukin -3, EGFP – enhanced green fluorescent protein.

Figure S1. Plasmid map of pSLIEW

Plasmid map drawn using Plasmapper (Dong, X, Stothard, P, Forsythe, I.J, and Wishart, D.S. "PlasMapper: a web server for drawing and auto-annotating plasmid maps" Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W660-4).

Figure S2. Removal of residual transduction capacity by washing

Supernatant harvested from transduced leukaemic blasts was put onto 293T cells grown in 6 well plates, and incubated overnight. The following day, supernatant was aspirated off and the 293T cells given fresh culture medium (DMEM, 10% v/v FCS, 2% v/v L-Glutamine, 1% v/v sodium pyruvate).

293T were assessed for transduction by expression of EGFP both by A) fluorescence microscopy - supernatant taken from transduced cells at day 3 and B) flow cytometry – supernatant was taken from transduced but unwashed leukaemic blasts at either day 3 (n=3) or day 4 (n=2) (mean + s.d.). Both demonstrate high residual transduction capacity. C) Effect of washing procedure described in supplementary methods above – by day 2, washing transduced leukaemic blasts has effectively removed residual transduction capacity from the supernatant, whilst the supernatant from unwashed blasts retains a substantial viral titre.

Figure S3. 3-Dimensional analysis of disease progression

The video demonstrates a 3dimensional reconstruction of the first generation mouse engrafted with transduced L578. Despite a relatively low level of transduction, 2%, bioluminescent imaging clearly demonstrates strong engraftment of the spleen and meninges and multiple sites of bone marrow engraftment, including the site of original transplantation, the right femur. The skeleton projected for orientation is representative and not derived directly from imaging of this animal.

Figure S4. Immunohistochemistry of organs engrafted with L578-SLIEW

Engrafted L578 leukaemic cells showed typical lymphoblastic morphology (a) and expressed characteristic proteins CD19 (b), CD10 (c) and TdT (d). There was widespread dissemination to multiple sites including spleen (e, i, m), kidney (f, j, n), liver (g, k, o) and meninges (h, l, p) as illustrated by staining with H&E (e-h) and for CD79a (i-l). EGFP positive cells are seen sparsely throughout the organs (m-p), consistent with the low levels of transduction in this experiment and Figure S5.

Figure S5. Organ specific expression of EGFP by L578 leukaemic blasts

Peripheral blood (sampled at week 13) and organs harvested from secondary recipients of L578 (M34, M35 and M36, week 17) were analysed by five-colour flow cytometry. Blasts taken from thetissuesof a single mouse show consistent levels of expression, demonstrating no systematic differential engraftment. N.B. Peripheral blood samples are based on low numbers of CD19+ cells (250-600 cells).

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