Figure 1.
Deficiency in p16Ink4a p19Arf cooperates with oncogenic KrasG12V to produce myeloid leukemia in NOD.SCID mice.
(A) Kaplan-Meier survival curve for NOD.SCID recipients after transplantation with C57BL/6 bone marrow (BM) cells modified by the indicated oncogenetic lesions. (B, C) Body weight and spleen weight analyses for NOD.SCID mice receiving p16p19−/−; Kras(G12V) BM cells via intra-venous transplantation at the time of sacrifice. Kras(G12V) and control groups were injected with cells and sacrificed for analysis at the same time points. (D) Hematoxylin & eosin stain of bone, spleen and liver from NOD.SCID leukemic mice, indicating metastasis of myeloid leukemia cells (60x). (E) Representative flow cytometry analysis of bone marrow from a NOD.SCID recipient of p16p19−/−; Kras(G12V)-GFP expressing cells. Data are shown as 2-parameter contour plots for Forward Scatter (FSC) or for the indicated cell surface makers. Plots at right show data for cells previously gated for viability (propidium iodide-, not shown) and GFP expression (leftmost plot).
Table 1.
Summary of intra-muscular injection in NOD.SCID mice.
Figure 2.
Identical leukemogenic system generates histiocytic sarcoma in the hind limb of NOD.SCID mice when injected into the gastrocnemius muscle.
(A) Kaplan-Meier curve showing the fraction tumor-free mice at the indicated time after transplant of p16p19−/−; Kras(G12V)-GFP with or without cardiotoxin (CTX) pre-injury, or of p16p19−/− ;Ctrl or WT; Kras(G12V) cells, with CTX pre-injury, into the muscles of NOD.SCID mice. (B, C) Body weight and spleen weight analyses at the time of sacrifice for NOD.SCID mice receiving p16p19−/−; Kras(G12V) or p16p19−/− ;Ctrl intra-muscular transplantation. Mice were sacrificed approximately 2 weeks after initial tumor detection. (D) Flow cytometry analysis of a representative tumor sample from a NOD.SCID mouse bearing an p16p19−/−; Kras(G12V)-GFP muscle tumor, revealing that most GFP+ tumor cells (gating shown on leftmost plot) are CD48hi, CD47hi, Mac1hi, but Gr1−/lo, B220−/lo, CD4−/lo, CD8−/lo, CD71−/lo and Ter119−/lo.
Figure 3.
Histopathological examination of histiocytic sarcoma tissue sections.
Hematoxylin & eosin staining of tumor (60x) indicating that myeloid cells comprise a majority of the tumor cells. Immunohistochemistry staining of tumor samples shows positive staining for GFP, Ki67, Mac2, and MPO, and negative staining of the non-myeloid hematopoietic lineage markers, B220 and Ter119 (40x).
Figure 4.
Phenotypic profiling of bone marrow and spleen cells indicates dissemination of tumor cells from hindlimb muscle into hematopoietic organs.
(A) H & E staining of bone, spleen and liver from recipients bearing primary myeloid tumor in the muscle, indicating aggressive infiltration of myeloid leukemia cells (60x) to distant anatomical locations. (B, C) Representative flow cytometry data demonstrating a similar immunophenotype of tumor cells in BM (B) and spleen (C) as that seen in the primary histiocytic sarcoma initiated in muscle (see Fig. 2D).
Figure 5.
Primary histiocytic sarcomas are serially transplantable in NOD.SCID mice.
(A) Schematic diagram showing experimental design for secondary transplantation. GFP+ cells were sorted from freshly isolated primary tumor, induced initially by transplantation of gene-modified bone marrow cells into the cardiotoxin pre-injured muscle of NOD.SCID recipients, and injected into gastrocnemius muscle of secondary NOD.SCID recipients at different cell dosages (100 000, 10 000, 2 000, or 200 cells) following cardiotoxin pre-injury. (B) H & E staining of tumor cells (60x) in skeletal muscle of a secondary recipient. Immunohistochemical staining of secondary tumors shows positive staining for the myeloid marker Mac2, and negative staining of B220 and Ter119 (40x). (C) H & E staining of liver and spleen from secondary recipient mouse, showing aggressive infiltration of myeloid leukemia cells (60x).
Figure 6.
Clonality analysis for genomic DNA samples extracted from primary and secondary histiocytic sarcomas and BM samples.
(A) Genomic DNA was extracted from primary and secondary tumors, and subjected to LM-PCR assay. Electrophoretic results of the final products are shown. Numbers at top of gel refer to sample numbers: No. 1 to 6 are primary tumors, No. 7 is a secondary tumor transplanted using GFP+ tumor cells from sample No. 4. All tumors possess an average of three integration sites, demonstrated by distinct numbers and sizes of bands (yellow numbers on the gel). HEK293T cells were transduced with the same virus and subject to LM-PCR as control. Size markers (100 bp ladder, size of individual bands indicated in red text) shown at left. (B) Genomic DNA was extracted from BM samples of the same set of NOD.SCID mice bearing primary and secondary tumors as analyzed in (A). DNA was subjected to LM-PCR assay. Electrophoretic results of the final products are shown. All BM samples possess an average of three to four integration sites. Bands unique of bone marrow samples are indicated by white arrowheads. Size markers (100 bp ladder) shown at left.