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Diverging fates of cells of origin in acute and chronic leukaemia.

Kovacic B, Hoelbl A, Litos G, Alacakaptan M, Schuster C, Fischhuber KM, Kerenyi MA, Stengl G, Moriggl R, Sexl V, Beug H - EMBO Mol Med (2012)

Bottom Line: During disease-maintenance, CML LT-HSCs persist to function as cancer stem cells (CSCs) that maintain leukaemia and require signalling by the transcription factor STAT5.In contrast, B-ALL LT-HSCs differentiate into CSCs that correspond to pro-B cells.This transition step requires a transient IL-7 signal and is lost in IL-7Rα-deficient cells.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Molecular Pathology (I.M.P.), Vienna, Austria. boris.kovacic@vetmeduni.ac.at

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Eradication of therapy-resistant BCR/ABLp210 CSCs by enforced differentiation and by STAT5-deletionCumulative cell number of BCR/ABLp210 LT-HSC and wildtype LT-HSCs grown under conditions as described in Supporting Information Fig S6. Wildtype LT-HSCs die at day 16.BCR/ABLp210 LT-HSCs induce a CML-like disease in secondary recipient mice (n = 3). Contributions of BCR/ABLp210+ LT-HSCs to myeloid and lymphoid lineages are indicated by FACS plots.BCR/ABLp210 LT-HSCs from long-term cultures were forced into terminal haematopoietic differentiation by addition of SCF, IL-3, IL-6, IL-11, GM-CSF, M-CSF and Epo to the serum-free medium. The FACS-plots on the left side show cells before addition of cytokines. Cells express myeloid, lymphoid and erythroid lineage markers such as Mac-1, CD19 and Ter119 after 7 days of cultivation (middle panels). FACS-plots from the peripheral blood of secondary transplants are depicted in the right panels. No leukaemia formation was observed (n = 4).Deletion of Stat5 from primary transplanted leukaemic LT-HSCs (BCR/ABLp210+ Mx1Cre+Stat5fl/fl LT-HSCs) abrogates leukaemia formation in 4:5 secondary recipients. The one moribund mouse showed strongly reduced leukaemic cell populations (bottom panel) as compared to control BCR/ABLp210+ Mx1Cre+Stat5+/+ LT-HSCs-transplanted mice.
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fig06: Eradication of therapy-resistant BCR/ABLp210 CSCs by enforced differentiation and by STAT5-deletionCumulative cell number of BCR/ABLp210 LT-HSC and wildtype LT-HSCs grown under conditions as described in Supporting Information Fig S6. Wildtype LT-HSCs die at day 16.BCR/ABLp210 LT-HSCs induce a CML-like disease in secondary recipient mice (n = 3). Contributions of BCR/ABLp210+ LT-HSCs to myeloid and lymphoid lineages are indicated by FACS plots.BCR/ABLp210 LT-HSCs from long-term cultures were forced into terminal haematopoietic differentiation by addition of SCF, IL-3, IL-6, IL-11, GM-CSF, M-CSF and Epo to the serum-free medium. The FACS-plots on the left side show cells before addition of cytokines. Cells express myeloid, lymphoid and erythroid lineage markers such as Mac-1, CD19 and Ter119 after 7 days of cultivation (middle panels). FACS-plots from the peripheral blood of secondary transplants are depicted in the right panels. No leukaemia formation was observed (n = 4).Deletion of Stat5 from primary transplanted leukaemic LT-HSCs (BCR/ABLp210+ Mx1Cre+Stat5fl/fl LT-HSCs) abrogates leukaemia formation in 4:5 secondary recipients. The one moribund mouse showed strongly reduced leukaemic cell populations (bottom panel) as compared to control BCR/ABLp210+ Mx1Cre+Stat5+/+ LT-HSCs-transplanted mice.

Mentions: Long-term cultivation of CSCs from CML has so far not been reported. When we cultivated ex vivo purified BCR/ABLp210+ LT-HSCs in serum-free medium plus defined growth factors (SCF/Tpo/IGF-II and FGF-1; Zhang & Lodish, 2005, 2008), the cells formed ‘cobblestone structures’ consisting of adherent and suspension cells (Supporting Information Fig S6C). Efficient cultivation was only achieved when the large (forward scatter, FSChigh) cells representing approximately 7–8% of the CSC-pool were re-plated (Supporting Information Fig S6C). Small (FSClow) BCR/ABLp210+ CSCs were unable to proliferate in vitro (Movies S1 and S2). Using this method, BCR/ABLp210+ CSCs were expanded >1014-fold within 67 days (Fig 6A). Wildtype LT-HSCs increased only about 100-fold and stopped to proliferate after 14 days. BCR/ABLp210+ CSCs expressed multiple markers indicative for HSCs (Supporting Information Tables SIIA and SIIB) and retained the ability to induce a fatal CML-like disease (n = 3) upon injection into lethally irradiated mice even after 103-fold expansion (Fig 6B). This CML recapitulated the phenotype observed in primary transplants and was dominated by Gr-1+/Mac-1+ leukaemic cells (Fig 6B). However, this leukaemogenic capacity was lost after 107-fold expansion (data not shown). We also tested whether our cultivation system extends to CSCs from caSTAT5+-induced disease. Purified caSTAT5+ LT-HSCs from moribund mice were maintained in SCF/Tpo/IGF-II and FGF-1 under serum-free conditions—enabling sustained proliferation (Supporting Information Fig S6D). Comparable to BCR/ABLp210 CSCs, the caSTAT5+ CSCs expressed HSC-surface markers in vitro (Supporting Information Tables SIIIA and SIIIB) and were able to induce CML in secondary transplants (data not shown). In summary, we show that BCR/ABLp210+ and caSTAT5+ CSCs may be expanded in vitro—retaining key features such as HSC surface marker expression, self-renewal and the ability to induce leukaemia upon transplantation.


Diverging fates of cells of origin in acute and chronic leukaemia.

Kovacic B, Hoelbl A, Litos G, Alacakaptan M, Schuster C, Fischhuber KM, Kerenyi MA, Stengl G, Moriggl R, Sexl V, Beug H - EMBO Mol Med (2012)

Eradication of therapy-resistant BCR/ABLp210 CSCs by enforced differentiation and by STAT5-deletionCumulative cell number of BCR/ABLp210 LT-HSC and wildtype LT-HSCs grown under conditions as described in Supporting Information Fig S6. Wildtype LT-HSCs die at day 16.BCR/ABLp210 LT-HSCs induce a CML-like disease in secondary recipient mice (n = 3). Contributions of BCR/ABLp210+ LT-HSCs to myeloid and lymphoid lineages are indicated by FACS plots.BCR/ABLp210 LT-HSCs from long-term cultures were forced into terminal haematopoietic differentiation by addition of SCF, IL-3, IL-6, IL-11, GM-CSF, M-CSF and Epo to the serum-free medium. The FACS-plots on the left side show cells before addition of cytokines. Cells express myeloid, lymphoid and erythroid lineage markers such as Mac-1, CD19 and Ter119 after 7 days of cultivation (middle panels). FACS-plots from the peripheral blood of secondary transplants are depicted in the right panels. No leukaemia formation was observed (n = 4).Deletion of Stat5 from primary transplanted leukaemic LT-HSCs (BCR/ABLp210+ Mx1Cre+Stat5fl/fl LT-HSCs) abrogates leukaemia formation in 4:5 secondary recipients. The one moribund mouse showed strongly reduced leukaemic cell populations (bottom panel) as compared to control BCR/ABLp210+ Mx1Cre+Stat5+/+ LT-HSCs-transplanted mice.
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fig06: Eradication of therapy-resistant BCR/ABLp210 CSCs by enforced differentiation and by STAT5-deletionCumulative cell number of BCR/ABLp210 LT-HSC and wildtype LT-HSCs grown under conditions as described in Supporting Information Fig S6. Wildtype LT-HSCs die at day 16.BCR/ABLp210 LT-HSCs induce a CML-like disease in secondary recipient mice (n = 3). Contributions of BCR/ABLp210+ LT-HSCs to myeloid and lymphoid lineages are indicated by FACS plots.BCR/ABLp210 LT-HSCs from long-term cultures were forced into terminal haematopoietic differentiation by addition of SCF, IL-3, IL-6, IL-11, GM-CSF, M-CSF and Epo to the serum-free medium. The FACS-plots on the left side show cells before addition of cytokines. Cells express myeloid, lymphoid and erythroid lineage markers such as Mac-1, CD19 and Ter119 after 7 days of cultivation (middle panels). FACS-plots from the peripheral blood of secondary transplants are depicted in the right panels. No leukaemia formation was observed (n = 4).Deletion of Stat5 from primary transplanted leukaemic LT-HSCs (BCR/ABLp210+ Mx1Cre+Stat5fl/fl LT-HSCs) abrogates leukaemia formation in 4:5 secondary recipients. The one moribund mouse showed strongly reduced leukaemic cell populations (bottom panel) as compared to control BCR/ABLp210+ Mx1Cre+Stat5+/+ LT-HSCs-transplanted mice.
Mentions: Long-term cultivation of CSCs from CML has so far not been reported. When we cultivated ex vivo purified BCR/ABLp210+ LT-HSCs in serum-free medium plus defined growth factors (SCF/Tpo/IGF-II and FGF-1; Zhang & Lodish, 2005, 2008), the cells formed ‘cobblestone structures’ consisting of adherent and suspension cells (Supporting Information Fig S6C). Efficient cultivation was only achieved when the large (forward scatter, FSChigh) cells representing approximately 7–8% of the CSC-pool were re-plated (Supporting Information Fig S6C). Small (FSClow) BCR/ABLp210+ CSCs were unable to proliferate in vitro (Movies S1 and S2). Using this method, BCR/ABLp210+ CSCs were expanded >1014-fold within 67 days (Fig 6A). Wildtype LT-HSCs increased only about 100-fold and stopped to proliferate after 14 days. BCR/ABLp210+ CSCs expressed multiple markers indicative for HSCs (Supporting Information Tables SIIA and SIIB) and retained the ability to induce a fatal CML-like disease (n = 3) upon injection into lethally irradiated mice even after 103-fold expansion (Fig 6B). This CML recapitulated the phenotype observed in primary transplants and was dominated by Gr-1+/Mac-1+ leukaemic cells (Fig 6B). However, this leukaemogenic capacity was lost after 107-fold expansion (data not shown). We also tested whether our cultivation system extends to CSCs from caSTAT5+-induced disease. Purified caSTAT5+ LT-HSCs from moribund mice were maintained in SCF/Tpo/IGF-II and FGF-1 under serum-free conditions—enabling sustained proliferation (Supporting Information Fig S6D). Comparable to BCR/ABLp210 CSCs, the caSTAT5+ CSCs expressed HSC-surface markers in vitro (Supporting Information Tables SIIIA and SIIIB) and were able to induce CML in secondary transplants (data not shown). In summary, we show that BCR/ABLp210+ and caSTAT5+ CSCs may be expanded in vitro—retaining key features such as HSC surface marker expression, self-renewal and the ability to induce leukaemia upon transplantation.

Bottom Line: During disease-maintenance, CML LT-HSCs persist to function as cancer stem cells (CSCs) that maintain leukaemia and require signalling by the transcription factor STAT5.In contrast, B-ALL LT-HSCs differentiate into CSCs that correspond to pro-B cells.This transition step requires a transient IL-7 signal and is lost in IL-7Rα-deficient cells.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Molecular Pathology (I.M.P.), Vienna, Austria. boris.kovacic@vetmeduni.ac.at

Show MeSH
Related in: MedlinePlus