Supplementary Materials
Supplementary Materials and Methods
Mice. All mouse lines were maintained in a pure C57BL/6 genetic background (>N10). The conditional LSL Kras G12D allele is described in (1). Mice bearing this allele were crossed to Mx1-Cre mice to generate mice carrying both alleles (LSL Kras G12D/+; Mx1-Cre/+). The CD44 knockout mice (CD44-/-) were obtained from Jackson Laboratories (Stock number 005085). LSL Kras G12D/+; CD44-/- was crossed to Mx1-Cre; CD44+/- to generate compound mice (LSL Kras G12D/+; Mx1-Cre; CD44-/-). The common b subunit knockout mice (bc-/-) were obtained from Jackson Laboratories (Stock number 005940). LSL Kras G12D/+; bc -/- was crossed to Mx1-Cre; bc -/- to generate compound mice (LSL Kras G12D/+; Mx1-Cre; bc -/-). Genotyping of Kras G12D, CD44, bc, and Mx1-Cre was done as previously described(2-4). CD45.1+ congenic recipient mice were purchased from NCI.
To induce Mx1-Cre expression, 5-7 week old mice were injected intraperitoneally with
250 mg of polyinosinic-polycytidylic acid (pI-pC; Sigma Aldrich, St. Louis, MO) every other day for two doses. All the experiments were performed two days after the 2nd injection of pI-pC unless specified. All experiments were conducted with the ethical approval of International Association for Assessment and Accreditation of Laboratory Animal Care at the University of Wisconsin, Madison.
Histopathology. Mouse organs were fixed in 10% neutral buffered formalin (Sigma-Aldrich) and further processed at the Histology Lab of the University of Wisconsin Carbone Cancer Center.
Complete blood count. Peripheral blood samples were collected in EDTA-coated tubes and complete blood count was performed at the Marshfield Laboratories.
Flow cytometric analysis of hematopoietic tissues. For lineage analysis of peripheral blood, bone marrow, spleen, and thymus tissues, flow cytometric analyses were performed as previously described (2) except that bone marrow and spleen were treated with Amonium Chloride Solution (StemCell Technologies) to lyse late erythroblasts and enucleated red blood cells prior to staining and flow analysis. Myeloid progenitors in bone marrow and spleen were analyzed as previously described (5). Hematopoietic stem cells (HSCs) in bone marrow and spleen were analyzed as described in (6). The stained cells were analyzed on a FACS Calibur or a LSRII (BD Biosciences).
Directly conjugated antibodies specific for the following surface antigens were purchased from eBioscience: CD45.2 (104), B220 (RA3-6B2), CD19 (eBio1D3), Thy1.2 (53-2.1), Mac-1 (M1/70), Gr-1 (RB6-8C5), CD4 (GK1.5), CD8 (53-6.7), CD3 (145-2C11), IgM (II/41), IL7Ra (A7R34), Sca-1 (D7), TER119(TER-119), CD34 (RAM34), CD44 (IM7). FcγRII/III (2.4G2) was purchased from BD Biosciences. CD25 (3C7) and CD150 (TC15-12F12.2) were purchased from Biolegend. Following biotin-conjugated antibodies were purchase from eBioscience: B220 (RA3-6B2), Gr-1 (RB6-8C5), CD8 (53-6.7). Following biotin-conjugated antibodies were purchased from BD Biosciences: CD19 (1D3), Mac-1 (M1/70), CD4 (RM4-5), CD3 (145-2C11), IgM (R6-60.2), IL7Ra (B12-1), TER119(TER-119).
Flow cytometric analysis of phospho-ERK1/2 and phospho-STAT5. Phosphorylated ERK1/2 and STAT5 were analyzed in defined Lin-/low c-Kit+ and Lin-/low c-Kit- cells essentially as previously described (7). Surface proteins were detected with FITC-conjugated antibodies (BD Biosciences unless specified) against B220 (6B2), Gr-1 (RB6-8C5), CD3 (17A2, Biolegend), CD4 (RM4-5), CD8 (53-6.7), and TER119, and PE-conjugated anti-CD117/c-Kit antibody (eBiosciences, San Diego, CA). p-ERK1/2 was detected by a primary antibody against pERK (Thr202/Tyr204; Cell signaling Technology) followed by APC conjugated donkey anti-rabbit F(ab’)2 fragment (Jackson ImmunoResearch). p-Stat5 (pY694) was detected by Alexa 647 conjugated primary antibody against phospho Stat5 (BD Biosciences).
Flow cytometric analysis of phospho-AKT. Phosphorylated AKT was analyzed in defined Lin-/low c-Kit+ cells essentially as previously described (7) with the following modifications. Briefly, cells were deprived of serum and cytokines for 2
hours before SCF stimulation. p-AKT was detected by a primary antibody against p-AKT (Ser473) (clone D9E, Cell signaling Technology) followed by APC conjugated donkey anti-rabbit F(ab’)2 fragment (Jackson ImmunoResearch). Cells were analyzed on a FACS Calibur (BD Biosciences). The data was analyzed using Flowjo software (Tree Star).
Transplantation of purified Kras G12D HSCs, CLPs, and thymic DN1_DN2 cells. Kras G12D HSCs were purified from bone marrow as [CD41 CD48 B220 Gr1 TER119]- CD150+ Sca1+cKit+ cells (6), CLPs were sorted from bone marrow and spleen as Lin- CD127+ Sca1lo c-Kitlo cells (8) , and thymic DN1_DN2 cells were purified from thymus as Lin- CD44+ cells. HSCs were transplanted into lethally irradiated mice as previously described (2). Because of extremely low frequency of CLPs, all the CLPs purified from bone marrow and spleen of a single mouse were transplanted together with 2 X 105 congeneic competitor/helper cells into one lethally irradiated mouse. Thymic DN1_DN2 cells were transplanted into sublethally irradiated recipient mice through intra-thymic injection (9) or co-transplanted with 2X105 competitor cells into lethally irradiated mice through retro-orbital injection.
Cell-cycle analysis of HSCs. Cell cycle analysis was performed essentially as described in (5). Briefly, cells were firstly stained with PE-Cy7 conjugated-antibodies against CD41, CD48, B220, TER119, and Gr1 (eBioscience), PE conjugated antibody against CD150 (Biolegend), PerCP-Cy5.5 conjugated antibody against Sca1 (eBioscience) and APC conjugated antibody against cKit (BD Biosciences). Cells were then fixed, permeabilized, and simultaneously stained with FITC conjugated antibody against Ki67 (BD Biosciences) and DAPI (Invitrogen). The stained cells were analyzed on a LSRII (BD Biosciences).
Apoptosis analysis of HSCs. Cell apoptosis analysis was performed essentially as described (10). Lineage markers (CD41, CD48, B220, TER119, and Gr1) were stained with PE-Cy7 conjugated antibodies (eBioscience). Cells were also simultaneously stained for FITC-AnnexinV (BD Biosciences), PE-CD150 (Biolegend), APC-cKit (BD Biosciences) and DAPI (Invitrogen). The stained cells were analyzed on a LSRII (BD Biosciences).
Mouse bone marrow transplantation. Bone marrow transplantation using total bone marrow cells was performed as described (7). When transplanting splenocytes, 1 X 106 or 5 X 106 splenocytes (CD45.2+) were mixed with 0.25 X 106 or 1 X 106 congenic bone marrow cells (CD45.1+) respectively and injected into individual lethally irradiated mice.
Characterization of Notch1 mutations. Genomic DNAs were isolated from thymus using the Puregene® Genomic DNA Purification Kit (Qiagen). Total RNAs were extracted from thymus using the RNeasy Mini Kit (Qiagen, Valencia, California). First strand cDNAs were synthesized using Super-Script First Strand Synthesis System (Invitrogen). Detection of Notch1 mutations in the exon 34 was performed as previously described (5). Type 1 deletion at the Notch1 locus was detected as described (11), while the corresponding wild-type allele was detected in genomic DNAs with primer WT-F (5’-ATAGCCAATCCATAGAGGGG
TC-3’) and Type 1 deletion reverse primer (5’CGTTTGGGTAGAAGAGATGCTTTAC-3’) under the same PCR condition for analyzing Type 1 deletion. Detection of Type 2 deletion was performed using cDNAs with primers T2F (5’-TTGCTCTGCCTAACGCTGCT -3’) and T2R(5’-AGGGAGAACTACTGG CTCCTCAAA-3’) according to the following conditions: 94℃ 2min, 35 cycles for 94℃ 30s, 58℃ 30s, 72℃ 1min.
Cell homing assay. Transient cell homing assay was performed essentially as described (12). Briefly, ten million CD45.2+ T-ALL tumor cells were incubated with 10 mg of either purified rat IgG (Sigma-Aldrich) or CD44 blocking antibody (BD Biosciences, clones IM7 and KM114) and 0.5µM of CFDA cell tracer dye per the manufacturer’s protocol (Invitrogen) (test cells). Ten million tumor cells were separately incubated with 10 mg of purified rat IgG alone (control cells). Test cells and control cells were mixed at 1:1 ratio and two million mixed cells were injected into lethally irradiated recipient mice (CD45.1+). Recipient mice were scarified two-hour after transplantation. Flow analysis was performed immediately before (input) and after (recovery) transplantation. Homing index was calculated as ratio of (test cells)recovery/(control cells)recovery to ratio of (test cells)input/(control cells)input .
Table S1. Only Kras G12D HSCs initiate T-ALL in recipient mice.
Cell Type / Injection dose / T-ALL mice/Recipient mice
HSCs / 3-20 cells / 29/59
CLPs / 1 donor:1 recipient (80-120 cells) / 0/6
Thymic
DN1_DN2 / 12,000-15,000 cells
(intra-thymic injection) / 0/24
20,000 cells
(retro-orbital injection) / 0/36
Supplemental Figure Legends
Figure S1. Loss of CD44 alleviates the acute MPN phenotypes in Kras G12D mice. (A) Genotyping analysis of genomic DNA to detect different alleles in representative control, Kras G12D, CD44-/-, and Kras G12D; CD44-/- mice. (B) Representative histologic H&E sections (10X, 60X) were shown for each genotype.
Figure S2. Kras G12D and Kras G12D; CD44-/- mice exhibit similar MPN phenotypes at the moribund stage. After pI-pC injections, control, Kras G12D, CD44-/-, and Kras G12D; CD44-/- mice were kept for an extended period of time until Kras G12D or Kras G12D; CD44-/- mice reached a moribund stage. (A) Splenomegaly in Kras G12D and Kras G12D; CD44-/- mice. Results are presented as scatter plots of the spleen weight of individual animals with mean ± s.d.. Student’s t-test was performed: * p < 0.05; n.s., not significant. (B) Flow cytometric analysis of bone marrow (BM), peripheral blood (PB), and spleen (SP) from representative mice using myeloid specific markers (Mac1 and Gr1). The percentages of cells enriched for monocytes (UL quadrant) and granulocytes (UR quadrant) are indicated beside the plots. Representative data from 3-5 mice in each group are shown. (C) Complete blood count of peripheral blood samples collected from control, Kras G12D and Kras G12D; CD44-/- mice. Asterisks indicate significant differences compared to the control group (p < 0.05).
Figure S3. SCF-evoked Akt activation is normal in Kras G12D and Kras G12D; CD44-/- cells. Five-seven week old mice were injected with pI-pC and sacrificed two days after the 2nd pI-pC injection. Total bone marrow cells were serum- and cytokine- starved for 2 hours and stimulated with various concentrations of SCF (0, 1, and 10 ng/ml) at 37°C for 10 minutes. Levels of phosphorylated AKT were measured using phospho-specific flow cytometry. Non-neutrophil bone marrow cells were gated for data analysis. Representative gating and plots of p-AKT are shown. Myeloid progenitors are enriched in c-Kit+ Lin-/low cells (R1). To quantify the activation of AKT, median intensities of p-AKT at different SCF concentrations in different groups of animals are compared to the control cells at 0 ng/ml, which is arbitrarily set at 1.
Figure S4. Apoptosis analysis of bone marrow HSCs. Five-seven week old mice were injected with pI-pC and sacrificed two days after the 2nd pI-pC injection. Bone marrow HSCs were defined as described in Figure 4. Representative contour plots of DAPI and Annexin V double stained HSCs are shown. The percentages of apoptotic cells (DAPI- Annexin V+ cells; LR quadrant) and dead cells (DAPI+ Annexin V+ cells; UR quadrant) are indicated on the plots.
Figure S5. Recipient mice with Kras G12D; CD44-/- cells develop similar hematopoietic malignancies as those with Kras G12D cells. (A) Flow cytometric analysis of total thymocytes of representative mice with control cells and TALL mice with Kras G12D/+ or Kras G12D; CD44-/- cells. (B) Flow cytometric analysis of peripheral blood of representative mice with control cells and MPN mice with Kras G12D/+ or Kras G12D; CD44-/- cells. The percentages of cells enriched for monocytes (UL quadrant) and granulocytes (UR quadrant) are indicated on the plots.
Figure S6. Notch1 mutations are detected in all T-ALL tumor samples with Kras G12D; CD44-/- cells. Genomic DNA and total RNA were extracted from T-ALL tumor samples with Kras G12D; CD44-/- cells and control thymocytes. (A) Genomic PCR analysis of Type 1 deletion and its corresponding wild-type (WT) allele at the Notch1 locus. (B) RT-PCR analysis of Type 2 deletion at the Notch1 locus. A T-ALL tumor sample with a known Type 2 deletion was used as a positive control (PC). (C) Sequence analysis of the exon 34 of Notch1. Sequencing results were aligned with WT sequence and inserted nucleotides near the PEST domain were listed.
Figure S7. CD44 is required for the homing of Kras G12D T-ALL cells to bone marrow. Recipient mice with Kras G12D cells developed T-ALL and were sacrificed at a moribund stage. Thymic lymphoma cells were isolated for the homing assay. (A) Experimental design for the short-term competitive homing of T-ALL cells. (B) Representative FACS plots show the relative input and recovery of transplanted cells. The percentages of controls cells (CD45.2+ CFDA-) and test cells (CD45.2+ CFDA+) are shown on the plots. (C) Homing index was calculated as described in the Materials and Methods. Results are shown as mean + s.d.. Student t test was performed. P values are shown on the plot.
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