In pediatric acute lymphoblastic leukemia (ALL), the most common hematologic malignancy in childhood, central nervous system (CNS) relapse is a major clinical problem, accounting for about one-third of the relapses.1 Since the early autopsy studies, CNS leukemia has been described primarily as a leptomeningeal disease which can be accompanied by infiltration of different brain parenchyma areas by ALL cells.2,3 The CNS is therefore considered to act as a sanctuary for ALL, but the specific neural microenvironments in which leukemic cells can stay for prolonged periods of time as extramedullary minimal residual disease and be responsible for CNS relapses are still poorly defined. Recently, we and others have reported that the choroid plexus stroma and the leptomeningeal stromal cell network are two of those neural microenvironments able to lodge ALL cells and promote their survival and acquisition of quiescence and chemoresistance. 4,5
Neurogenic niches, such as the subventricular zone (SVZ) and the subgranular zone, are areas of the brain in which neurogenesis takes place throughout life. Concretely, the SVZ is located along the walls of the lateral brain ventricles and represents the largest neurogenic niche in the postnatal and adult mammalian brain. Neural stem cells residing in the SVZ divide slowly and give rise to rapidly proliferating cells, called transit amplifying progenitors, which then differentiate to neuroblasts. These immature neuronal progenitors further migrate along a pathway, called the rostral migratory stream, towards the olfactory bulb where they differentiate into olfactory interneurons and integrate in the existing neuronal circuitry involved in odor discrimination.6 Nevertheless, the neurogenic niches, apart from their ability to support and maintain neural stem cells, can also serve as refuge for neoplastic cells, and the migratory mechanisms of neural stem cells can be utilized by tumor cells;7 therefore, in this study we investigated whether the SVZ can also harbor ALL cells.
A xenograft model of non-obese diabetic/severe combined immunodeficiency/IL-2Rgnull (NSG) mice injected with the Nalm-6 human pre-B ALL cell line was used to analyze the presence of leukemia cells in the brain SVZ. When symptoms of CNS involvement (such as hind limb paralysis) appeared, animals were deeply anesthetized and transcardially perfused immediately before the SVZ were carefully dissected and dissociated. Flow cytometry analysis of cells recovered from dissociated SVZ revealed that all mice with leukemic infiltration in the CNS showed CD19+ human blasts in this brain location (Figure 1A), and these ALL cells could represent up to 30% of total leukemic cells invading the nervous parenchyma (Figure 1B). Similar results were obtained with mice xenografted with other ALL cell lines, REH and RS4;11 (Figure 1C). However, no correlation was seen between the degree of leukemic infiltration in brain parenchyma and the proportion of ALL cells present in the SVZ. Immunofluorescence studies in brain cryosections from xenografted mice showed that leukemic cells could be seen, apart from in the SVZ niche, also along the rostral migratory stream (Online Supplementary Figure S1). These data indicate that the SVZ can provide a favorable microenvironment in which ALL cells can survive and be maintained over time. Supporting this, the study of the leukemia proliferation rate using Ki-67 staining showed that ALL cells found in the SVZ niche exhibit a much lower proliferative activity than those leukemic cells isolated from the meninges (Figure 1D). Furthermore, leukemic cells infiltrating the SVZ niche were shown to have higher chemoresistance after methotrexate treatment of xenografted mice (Figure 1E).
The above results showed that leukemic invasion of the SVZ neurogenic niche is a common event in the xenograft model of ALL, so we analyzed the effects of this infiltration on the differentiation of neural stem cells. The proportion of the different SVZ populations was determined by flow cytometry using a combination of multiple specific cell markers, as we previously described.8 Non-neurogenic cells were first discarded from the study, and the remaining neurogenic lineage cell pool was subdivided according to the expression of the glial marker GLAST, the neuroblast marker CD24, the tetraspanin CD9 and the proliferation-associated receptor EGFR. The population of neural stem cells was defined as GLAST+CD24-/lowCD9high and further classified by EGFR expression and GLAST intensity into quiescent, primed quiescent and activated neural stem cells. Transit amplifying progenitors were defined as GLAST-CD24- /lowEGFR+ cells, and the GLAST-CD24high population included EGFR+ proliferating (NB1 or early) and EGFR-/low migrating (NB2 or late) neuroblasts. As can be seen in Figure 2A, the percentage of total neural stem cells was notably increased in xenografted mice, with quiescent neural stem cells being the main subset responsible for this rise. Concomitantly, the proportion of transit amplifying progenitors and late neuro-blasts was reduced in these animals. These results suggest that SVZ neurogenesis is impaired in leukemia-bearing mice at the expense of an increase in quiescence, and the effect appears to be a direct consequence of the leukemic cell infiltration in the SVZ since the most affected animals were those showing the highest numbers of CD19+ cells in the neurogenic niche. Figure 2B shows that the percentages of CD19+ leukemia cells correlated directly with the accumulation of quiescent neural stem cells, and inversely with the proportions of late neuroblasts (Figure 2C). In line with these data, and since the generation of new olfactory bulb neurons from the SVZ is required for novel odor discrimination,9 xenografted mice displayed altered olfactory discrimination capacities (Figure 2D).
To analyze the effects of leukemia on neural precursors directly, we first generated SVZ neurospheres, floating cellular aggregates clonally derived from neurosphere-initiating neural stem cells which constitute an ideal system to evaluate modifications in proliferation and self-renewal. Single cells dissociated from neurospheres were either cultured with medium conditioned by leukemic cells or co-cultured with ALL cells using transwell inserts. In both cases, although no change in the number of new neurospheres was found after 10 days (Figure 3A), a significant reduction in neurosphere sizes could be clearly observed (Figure 3B), suggesting that leukemia-derived factors limit growth but not survival of neurosphere cells. In agreement, the inhibition of the expansion potential of neurospheres in the presence of leukemia cells was detected throughout the culture period using MTS proliferation assays (Figure 3C). To analyze whether ALLmediated effects included effects on self-renewal, cells obtained from neurospheres that had been grown in the presence of soluble factors secreted by leukemic blasts were re-plated in fresh growth medium without leukemia-derived factors and neurosphere formation was evaluated. In these cultures, the numbers of secondary neurospheres were not altered but significant changes in sphere diameters could be newly detected, indicating that leukemia cells can reduce activation without altering self-renewal (Figure 3D, E).
ALL cells have been reported to be able to induce a proinflammatory microenvironment in different locations.4,10 The expression of pro-inflammatory factors was therefore analyzed in the SVZ of healthy and leukemic mice. As shown in Figure 3F, the levels of IL-1b, IL-6 and TNFα cytokines as well as CCL2 and CXCL10 chemokines were notably upregulated in the leukemia-invaded SVZ niches. All these inflammatory mediators have been described as negative regulators of neurogenesis.11 However, we have recently reported that TNF-a, which underwent one of the highest increases in expression, reduces neuroblast generation because it induces a transient activation of neural stem cells followed by their entry into quiescence.8 Transgenic mice overexpressing IL-6 in astrocytes exhibit reduced cycling of neural stem cells in the subgranular zone niche, suggesting that it acts as a negative regulator of proliferation,12 and IL-1b is reportedly secreted by choroid plexus cells to the cerebrospinal fluid and induces the upregulation of VCAM-1 levels in SVZ neural stem cells, reducing their proliferation and preventing lineage progression.13 Importantly, the levels of IL-1b, IL-6, TNF-a, CCL2 and CXCL10 have also been described to be increased in blood and cerebrospinal fluid of ALL patients, promoting the survival and quiescence of leukemic cells.14,15
Taken together, the results of the present study show that infiltration of the SVZ may be a common event in childhood ALL with CNS involvement, suggesting that SVZ is a sanctuary in which ALL cells could lodge, survive for prolonged periods of time and be responsible for future CNS relapses. Our results also show that leukemic infiltration of the SVZ neurogenic niche impairs neurogenesis, which likely leads to deleterious effects on brain functions. It is important to note that in human infants and young children not all neuroblasts born in the SVZ migrate to the olfactory bulb, but many of them migrate into the ventromedial prefrontal cortex as well as multiple regions of the frontal cortex, such as the cingulate gyrus.16,17 The late incorporation of inhibitory interneurons into those regions of the developing human brain has been proposed to constitute a mechanism of delayed postnatal plasticity and, therefore, injuries affecting neuronal recruitment during this period could contribute to neurocognitive deficits and sensorimotor disturbances, 16,17 such as those reported in ALL patients at diagnosis, before treatment initiation.18
- Received June 8, 2021
- Accepted January 7, 2022
Disclosures: MR has received a research grant from Orgenesis Inc.
Contributions: LMFS performed experiments, acquired the data, analyzed results, interpreted data and wrote the manuscript; GB, BMP and POS performed experiments, acquired the data and analyzed results; MR and IF co-designed the study and interpreted data; AVa and AVi conceived and designed the research, interpreted data and wrote the manuscript.
this work was supported by grants RTI2018-093899-BI00 and SAF2017-86690-R (Spanish Ministry of Economy and Competitiveness), RD16/0011/0002 and CB06/05/0086 (Institute of Health Carlos III, Spain), B2017/BMD-3692 AvanCell-CM (Community of Madrid), and Beca I-UnoEntreCienMil (Uno Entre Cien Mil Foundation). LMFS was supported by a pre doctoral fellowship (CT45/15 CT46/15) from the Complutense University of Madrid. POS is supported by a pre-doctoral fellowship (CT63/19- CT64/19) from the Complutense University.
- Frishman-Levy L, Izraeli S.. Advances in understanding the pathogenesis of CNS acute lymphoblastic leukaemia and potential for therapy. Br J Haematol. 2017; 176(2):157-167. https://doi.org/10.1111/bjh.14411PubMedGoogle Scholar
- Thomas LB. Pathology of leukemia in the brain and meninges: postmortem studies of patients with acute leukemia and of mice given inoculations of L1210 leukemia. Cancer Res. 1965; 25(9):1555-1571. Google Scholar
- Kinjyo I, Bragin D, Grattan R, Winter SS, Wilson BS. Leukemiaderived exosomes and cytokines pave the way for entry into the brain. J Leukoc Biol. 2019; 105(4):741-753. https://doi.org/10.1002/JLB.3A0218-054RPubMedPubMed CentralGoogle Scholar
- Fernandez-Sevilla LM, Valencia J, Flores-Villalobos MA. The choroid plexus stroma constitutes a sanctuary for paediatric B-cell precursor acute lymphoblastic leukaemia in the central nervous system. J Pathol. 2020; 252(2):189-200. https://doi.org/10.1002/path.5510PubMedPubMed CentralGoogle Scholar
- Jonart LM, Ebadi M, Basile P, Johnson K, Makori J, Gordon PM. Disrupting the leukemia niche in the central nervous system attenuates leukemia chemoresistance. Haematologica. 2020; 105(8):2130-2140. https://doi.org/10.3324/haematol.2019.230334PubMedPubMed CentralGoogle Scholar
- Fontán-Lozano A, Morcuende S, Davis-López de Carrizosa MA. To become or not to become tumorigenic: subventricular zone versus hippocampal neural stem cells. Front Oncol. 2020; 10:602217. https://doi.org/10.3389/fonc.2020.602217PubMedPubMed CentralGoogle Scholar
- Sinnaeve J, Mobley BC, Ihrie RA. Space invaders: brain tumor exploitation of the stem cell niche. Am J Pathol. 2018; 188(1):29-38. https://doi.org/10.1016/j.ajpath.2017.08.029PubMedPubMed CentralGoogle Scholar
- Belenguer G, Duart-Abadia P, Jordan-Pla A. Adult neural stem cells are alerted by systemic inflammation through TNF-alpha receptor signaling. Cell Stem Cell. 2021; 28(2):285-299. https://doi.org/10.1016/j.stem.2020.10.016PubMedGoogle Scholar
- Alonso SB, Reinert JK, Marichal N. An increase in neural stem cells and olfactory bulb adult neurogenesis improves discrimination of highly similar odorants. EMBO J. 2019; 38(6):e98791. https://doi.org/10.15252/embj.201798791PubMedPubMed CentralGoogle Scholar
- Vilchis-Ordonez A, Contreras-Quiroz A, Vadillo E. Bone marrow cells in acute lymphoblastic leukemia create a proinflammatory microenvironment influencing normal hematopoietic differentiation fates. Biomed Res Int. 2015; 2015:386165. https://doi.org/10.1155/2015/386165PubMedPubMed CentralGoogle Scholar
- Voloboueva LA, Giffard RG. Inflammation, mitochondria, and the inhibition of adult neurogenesis. J Neurosci Res. 2011; 89(12):1989-1996. https://doi.org/10.1002/jnr.22768PubMedPubMed CentralGoogle Scholar
- Brett FM, Mizisin AP, Powell HC, Campbell IL. Evolution of neuropathologic abnormalities associated with blood-brain barrier breakdown in transgenic mice expressing interleukin-6 in astrocytes. J Neuropathol Exp Neurol. 1995; 54(6):766-775. https://doi.org/10.1097/00005072-199511000-00003PubMedGoogle Scholar
- Kokovay E, Wang Y, Kusek G. VCAM1 is essential to maintain the structure of the SVZ niche and acts as an environmental sensor to regulate SVZ lineage progression. Cell Stem Cell. 2012; 11(2):220-230. https://doi.org/10.1016/j.stem.2012.06.016PubMedGoogle Scholar
- Gomez AM, Martinez C, Gonzalez M. Chemokines and relapses in childhood acute lymphoblastic leukemia: a role in migration and in resistance to antileukemic drugs. Blood Cells Mol Dis. 2015; 55(3):220-227. https://doi.org/10.1016/j.bcmd.2015.07.001PubMedGoogle Scholar
- Perez-Figueroa E, Sanchez-Cuaxospa M, Martinez-Soto KA. Strong inflammatory response and Th1-polarization profile in children with acute lymphoblastic leukemia without apparent infection. Oncol Rep. 2016; 35(5):2699-2706. https://doi.org/10.3892/or.2016.4657PubMedGoogle Scholar
- Sanai N, Nguyen T, Ihrie RA. Corridors of migrating neurons in the human brain and their decline during infancy. Nature. 2011; 478(7369):382-386. https://doi.org/10.1038/nature10487PubMedPubMed CentralGoogle Scholar
- Paredes MF, James D, Gil-Perotin S. Extensive migration of young neurons into the infant human frontal lobe. Science. 2016; 354(6308):aaf7073. https://doi.org/10.1126/science.aaf7073PubMedPubMed CentralGoogle Scholar
- Reinders-Messelink H, Schoemaker M, Snijders T. Motor performance of children during treatment for acute lymphoblastic leukemia. Med Pediatr Oncol. 1999; 33(6):545-550. https://doi.org/10.1002/(SICI)1096-911X(199912)33:6<545::AID-MPO4>3.0.CO;2-YGoogle Scholar
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