American Society of Hematology

Figure 2

$Figure 2. Lymphoma SP cells are enriched in clonogenic capacity and represent a transient phenotype. (A) Colony formation from purified lymphoma SP cells (CFUs), compared with no or only few colonies from corresponding non-SP cell preparations (representative example for Balm-3 survey, upper row; microscopic view, lower row). (B) For 5 cell lines, CFU activity was significantly enriched in the SP compared with non-SP cells (unpaired 2-sided t test; data for each individual cell line; see supplemental Figure 2). (C-D) In vivo, 0.5 × 106 purified SP cells generated tumors within 8 days in a modified CAM assay in ovo, compared with no or significantly smaller tumors from equal amounts of non-SP cells (C, representative examples of tumors; D, tumor mass; unpaired 2-sided Student t test, ****P < .001; n = 19; cell line OCI Ly3). (E) After admixture of 4% GFP-positive SP cells to 96% unlabeled non-SP cells, contribution of GFP-positive cells to the whole tumor were measured by flow cytometry and microscopy in vitro and in vivo. In the course of tumor expansion (left lower row; expansion of all cells ×106), the proportion of GFP-positive cells and of non-SP cells expanded, with the emergence of GFP-negative SP cells (upper row, example of flow cytometric GFP analysis of SP and non-SP at day 19; right lower row, representative results of 3 independent experiments). (F) After implantation of mixed cultures into the egg, GFP-positive SP cells contributed to both SP and non-SP cell fractions at day 8 of tumor growth in vivo, again with progeny from GFP-negative non-SP cells to both populations at day 8 in the cell line OCI Ly3 and OCI Ly1 (F, n = 2 each). (G) In the OCI Ly3 tumors after in ovo passaging, GFP-positive cells (arrowheads) were distributed evenly between progeny of non-SP cells throughout the tumor tissue (immunofluorescence staining with an anti-GFP antibody, n = 10; enlargement of insert ×8). (H) Collectively, whereas isolated non-SP cells died off in culture or in the host (A) and isolated SP cells formed colonies (B), in mixed cultures both SP and non-SP cells contributed to tumor propagation (C).$

Lymphoma SP cells are enriched in clonogenic capacity and represent a transient phenotype. (A) Colony formation from purified lymphoma SP cells (CFUs), compared with no or only few colonies from corresponding non-SP cell preparations (representative example for Balm-3 survey, upper row; microscopic view, lower row). (B) For 5 cell lines, CFU activity was significantly enriched in the SP compared with non-SP cells (unpaired 2-sided t test; data for each individual cell line; see supplemental Figure 2). (C-D) In vivo, 0.5 × 10⁶ purified SP cells generated tumors within 8 days in a modified CAM assay in ovo, compared with no or significantly smaller tumors from equal amounts of non-SP cells (C, representative examples of tumors; D, tumor mass; unpaired 2-sided Student t test, ****P < .001; n = 19; cell line OCI Ly3). (E) After admixture of 4% GFP-positive SP cells to 96% unlabeled non-SP cells, contribution of GFP-positive cells to the whole tumor were measured by flow cytometry and microscopy in vitro and in vivo. In the course of tumor expansion (left lower row; expansion of all cells ×10⁶), the proportion of GFP-positive cells and of non-SP cells expanded, with the emergence of GFP-negative SP cells (upper row, example of flow cytometric GFP analysis of SP and non-SP at day 19; right lower row, representative results of 3 independent experiments). (F) After implantation of mixed cultures into the egg, GFP-positive SP cells contributed to both SP and non-SP cell fractions at day 8 of tumor growth in vivo, again with progeny from GFP-negative non-SP cells to both populations at day 8 in the cell line OCI Ly3 and OCI Ly1 (F, n = 2 each). (G) In the OCI Ly3 tumors after in ovo passaging, GFP-positive cells (arrowheads) were distributed evenly between progeny of non-SP cells throughout the tumor tissue (immunofluorescence staining with an anti-GFP antibody, n = 10; enlargement of insert ×8). (H) Collectively, whereas isolated non-SP cells died off in culture or in the host (A) and isolated SP cells formed colonies (B), in mixed cultures both SP and non-SP cells contributed to tumor propagation (C).

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