Early B220^low cells are clonal precursors of postregression tumor cells. (A) Smear preparation of blood of pre- and postregression Eμ-myc mice. Giemsa-stained blood smears from WT revealed lymphocytes with small nuclei (upper left) while those from Eμ-myc mice at <41 days of age (upper middle) and >65 days of age (upper right) showed atypical lymphoid cells with enlarged and irregular nuclei. Arrows indicate lymphoid cells. Giemsa-stained blood smears were analyzed by light microscopy at original magnification ×600. B220^low cells of WT (filled gray), preregression Eμ-myc mice (dashed line), or postregression Eμ-myc mice (black line) were assessed for size by forward light scatter (lower left panel) and for intracellular KI-67 expression (lower right) by flow cytometry. (B) Expression levels of cell-cycle–related proteins in splenic B220⁺ cells of Eμ-myc mice at 23 and 68 ± 2 days of age and B220⁺ cells of WT mice at 23 days of age. Quantification of bands is shown in lower panel. Expression levels were normalized to WT levels. Shown is 1 of 3 representative experiments. (C) Splenic B220^low cells isolated from Eμ-myc mice at 37 and 81 ± 2 days of age were analyzed for malignant transformation by transfer of 5 × 10⁶ B220⁺ cells into SCID IL-2Rγ_c^−/− mice. BC2 cells, a cell line derived from Eμ-myc mice, were transferred as a positive control. Kaplan-Meier survival curve of SCID IL-2Rγ_c^−/− (left panel) or WT mice (right panel) after transfer of B220⁺ cells (top panel). In a separate experiment, 5 × 10⁶ splenic B220⁺ cells of individual Eμ-myc mice before 41 and after 65 days of age were transferred into different SCID IL-2Rγ_c^−/− mice. The number of B220^low cells in the blood of SCID IL-2Rγ_c^−/− mice 77 days after transfer was determined as outlined in Figure 1C. Mean number of B220^low cells in the blood of SCID IL-2Rγ_c^−/− mice is represented by a black line (bottom). (D) V-J junction sequences of the immunoglobulin κ light chain of pre- and postregression tumors. Complimentary DNA samples of preregression and postregression Eμ-myc mice (n = 5) were amplified by PCR using primers hybridizing to related sets of V regions of the κ light-chain genes in combination with a primer specific for the constant region (see “Methods” and supplemental Figure 4). Identical V-J junction sequences of pre- and postregression tumors of 3 out of 5 mice are shown. No successful V-J recombinations were found in the remaining 2 mice. Successful V-J junctions were compared with germline sequence of C57BL/6 mice using IMGT/V-QUEST (http://www.imgt.org). Blue letters show the V region, red letters indicate the J region, and black letters show mutations. (E) Complimentary DNA of blood tumor cells of pre- and postregression Eμ-myc mice was amplified using primers specific for different variable regions of the κ light chain and a common primer specific for the constant region of the light chain according to the manufacturer’s instructions (Progen Biotechnik GmbH, Heidelberg, Germany). d, day; E, tumors before 41 days of age; FSC, forward light scatter; L, tumors after 65 days of age; RSS, recombination signal sequences.