Contributions
Abstract: EP324
Type: E-Poster Presentation
Session title: Acute lymphoblastic leukemia - Biology & Translational Research
Background
B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells that are present in the bone marrow microenvironment are able to hijack the normal hematopoietic stem cell niches to create a leukemic niche. The importance of this microenvironment for leukemic cells is demonstrated by the protection that the niche provides against chemotherapeutic agents. Patient derived mesenchymal stromal cells (MSCs) mimic this protective effect in vitro. Unraveling the mechanism of protection is important to provide potential novel therapeutics. Therefore, our research is focused on the interaction between ALL cells and MSCs.
Aims
We aimed to determine gene expression changes in ALL cells and MSCs after co-culture compared to mono-culture, and to investigate which cyto/chemokines are differentially secreted upon contact between ALL cells and MSCs.
Methods
We performed mono- and co-cultures of primary MSCs and ALL cells for 40 hours to determine gene expression changes. Viable cells were sorted by FACS and RNA was isolated. Total-RNA sequencing data (Illumina) were analyzed using R. Supernatant was saved to determine cyto/chemokine profiles by Luminex technology, and to investigate the effect of these cyto/chemokines on the survival and migration of the leukemic cells. Migration experiments using transwells (3.0µm pores) were performed to determine the level of ALL migration towards chemokines of interest. Moreover, using lentivirus, knockdowns (KD) of genes of interest were performed in MSCs. KD efficiency was tested using Taqman qPCR. Subsequently, co-cultures of ALL cells and KD- or control-MSCs were performed to study the role these genes in ALL survival.
Results
RNA sequencing data from 15 independent co-culture experiments revealed that interferon (IFN)-related genes, such as IFI6, MX1, IFI27, and OAS1, were 2.5 to 3.1-fold upregulated in the MSCs after co-culture with ALL compared to MSC mono-culture. The type of upregulated pro-inflammatory genes and amount of upregulation varied between ALL patients. However, the observed changes were always similar when an ALL case was co-cultured with different MSC samples, suggesting that the changes are induced by the leukemic cells. Survival benefit (0.3 - 37.9%) was observed in ALL cells after co-culture with MSCs compared to ALL mono-culture. Co-cultures of KD-MSCs and ALL cells showed that ALL cell viability was affected upon KD of some IFN-related genes. The effect of the number of viable MSCs in these cultures is currently being addressed. Moreover, pro-inflammatory cytokines, and migration-related chemokines such as CCL2, CXCL8, and IP10 were upregulated after co-culture compared to the sum of the separate ALL and MSC mono-cultures. A gradient of IP10 in transwell experiments showed that this chemokine did not enhance primary ALL cell migration, suggesting that the ALL-induced secretion of IP10 serves a different role in BCP-ALL. The role of IP10 and other cyto/chemokines in immune regulation at the time of overt leukemia is part of ongoing studies.
Conclusion
Our data show that IFN-related genes, pro-inflammatory cytokines and migration-related chemokines become upregulated in bone marrow MSCs upon exposure to ALL. These induced changes may be important for ALL cell survival, affecting the mobility of other immune cells, and/or ensure that leukemic cells remain in close contact with MSCs. We postulate that interference with these affected genes and cyto/chemokines may disrupt the direct contact between leukemic cells and their niche and may provide an alternative way to eliminate leukemic cells more efficaciously.
Keyword(s): B cell acute lymphoblastic leukemia, Interferon, Mesenchymal stem cell
Abstract: EP324
Type: E-Poster Presentation
Session title: Acute lymphoblastic leukemia - Biology & Translational Research
Background
B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells that are present in the bone marrow microenvironment are able to hijack the normal hematopoietic stem cell niches to create a leukemic niche. The importance of this microenvironment for leukemic cells is demonstrated by the protection that the niche provides against chemotherapeutic agents. Patient derived mesenchymal stromal cells (MSCs) mimic this protective effect in vitro. Unraveling the mechanism of protection is important to provide potential novel therapeutics. Therefore, our research is focused on the interaction between ALL cells and MSCs.
Aims
We aimed to determine gene expression changes in ALL cells and MSCs after co-culture compared to mono-culture, and to investigate which cyto/chemokines are differentially secreted upon contact between ALL cells and MSCs.
Methods
We performed mono- and co-cultures of primary MSCs and ALL cells for 40 hours to determine gene expression changes. Viable cells were sorted by FACS and RNA was isolated. Total-RNA sequencing data (Illumina) were analyzed using R. Supernatant was saved to determine cyto/chemokine profiles by Luminex technology, and to investigate the effect of these cyto/chemokines on the survival and migration of the leukemic cells. Migration experiments using transwells (3.0µm pores) were performed to determine the level of ALL migration towards chemokines of interest. Moreover, using lentivirus, knockdowns (KD) of genes of interest were performed in MSCs. KD efficiency was tested using Taqman qPCR. Subsequently, co-cultures of ALL cells and KD- or control-MSCs were performed to study the role these genes in ALL survival.
Results
RNA sequencing data from 15 independent co-culture experiments revealed that interferon (IFN)-related genes, such as IFI6, MX1, IFI27, and OAS1, were 2.5 to 3.1-fold upregulated in the MSCs after co-culture with ALL compared to MSC mono-culture. The type of upregulated pro-inflammatory genes and amount of upregulation varied between ALL patients. However, the observed changes were always similar when an ALL case was co-cultured with different MSC samples, suggesting that the changes are induced by the leukemic cells. Survival benefit (0.3 - 37.9%) was observed in ALL cells after co-culture with MSCs compared to ALL mono-culture. Co-cultures of KD-MSCs and ALL cells showed that ALL cell viability was affected upon KD of some IFN-related genes. The effect of the number of viable MSCs in these cultures is currently being addressed. Moreover, pro-inflammatory cytokines, and migration-related chemokines such as CCL2, CXCL8, and IP10 were upregulated after co-culture compared to the sum of the separate ALL and MSC mono-cultures. A gradient of IP10 in transwell experiments showed that this chemokine did not enhance primary ALL cell migration, suggesting that the ALL-induced secretion of IP10 serves a different role in BCP-ALL. The role of IP10 and other cyto/chemokines in immune regulation at the time of overt leukemia is part of ongoing studies.
Conclusion
Our data show that IFN-related genes, pro-inflammatory cytokines and migration-related chemokines become upregulated in bone marrow MSCs upon exposure to ALL. These induced changes may be important for ALL cell survival, affecting the mobility of other immune cells, and/or ensure that leukemic cells remain in close contact with MSCs. We postulate that interference with these affected genes and cyto/chemokines may disrupt the direct contact between leukemic cells and their niche and may provide an alternative way to eliminate leukemic cells more efficaciously.
Keyword(s): B cell acute lymphoblastic leukemia, Interferon, Mesenchymal stem cell