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It is widely accepted that complex interactions between cancer cells and their surrounding microenvironment contribute to disease development, chemo-resistance and disease relapse. In light of this observed interdependency, novel therapeutic interventions that target specific cancer stroma cell lineages and their interactions are being sought. To this end, we studied a mouse model of human T cell acute lymphoblastic leukaemia (T-ALL) and used intravital microscopy to monitor the progression of disease within the bone marrow at both the tissue-wide and single cell level over time, from bone marrow seeding to development/selection of chemo-resistance. We observed highly dynamic cellular interactions and promiscuous distribution of leukaemia cells that migrated across the bone marrow, without showing any preferential association with bone marrow sub-compartments. Unexpectedly, this behaviour was maintained throughout disease development, from the earliest bone marrow seeding to response and resistance to chemotherapy. Our results reveal that T-ALL cells do not depend on specific bone marrow microenvironments for propagation of disease, nor for the selection of chemo-resistant clones, suggesting a stochastic mechanism underlies these processes. Yet, while T-ALL infiltration and progression are independent of the stroma, accumulated disease burden leads to rapid, selective remodelling of the endosteal space, resulting in a complete loss of mature osteoblastic cells whilst perivascular cells are maintained. This outcome leads to a shift in the balance of endogenous bone marrow stroma, towards a composition associated with less efficient haematopoietic stem cell function1. This novel, dynamic analysis of T-ALL interactions with the bone marrow microenvironment in vivo, supported by evidence from human T-ALL samples, highlights that future therapeutic interventions should target the migration and promiscuous interactions of cancer cells with the surrounding microenvironment, rather than specific bone marrow stroma, in order to combat the invasion by and survival of chemo-resistant T-ALL cells.
The importance of cancer cell interactions with their surrounding microenvironment has gained increased attention due to the hypothesis that specific supportive cells may regulate quiescence, survival and self-renewal of cancer cells themselves. This relationship may underlie a critical mechanism that facilitates both the initiation of disease and chemo-resistance. Leukaemia develops within the bone marrow (BM), where it has been suggested to take part in complex crosstalk that in some cases results in microenvironment remodelling2–8. Therapeutic targeting of leukaemia-supportive niches has been proposed9,10, therefore it is critical that we understand both the spatial and kinetic nature of leukaemia-BM interactions. However, our current knowledge of leukaemia biology is predominantly derived from ex vivo flow cytometric analysis, and static images that cannot capture information on the location and dynamics of leukaemia interactions with BM structures and cells over time.
We studied a Notch-driven mouse model of T cell acute lymphoblastic leukaemia (T-ALL), which recapitulates human disease both phenotypically (Extended Data Fig. 1) and genetically11,12. 25% paediatric and 40% adult T-ALL patients develop aggressive relapsed disease originating from chemo-resistant clones13. Thus, there is a pressing need to understand if T-ALL cells migrate to, and interact with particular BM stroma during the propagation of disease and/or selection of chemo-resistance, or if T-ALL can remodel the BM microenvironment in its favour.
To address these questions, we monitored leukaemia growth in mouse calvarium bone marrow by intravital microscopy14–16. We used a tile-based imaging approach akin to Google Earth that allows tissue-wide visualisation of heterogeneous BM microenvironments (Fig. 1a, b) whilst maintaining resolution that permits measurement of single leukaemia cell interactions with BM cells and structures by time-lapse microscopy15 (Fig. 1c and Supplementary Video 1). To systematically characterise T-ALL interactions in vivo, we injected transformed leukaemic cells isolated from primary hosts into secondary recipients, and observed highly synchronous disease progression (Fig. 1d and Extended Data Fig. 1). In secondary recipients, T-ALL preferentially infiltrated the BM before expansion to peripheral lymphoid organs (Extended Data Fig. 1). This is consistent with expression of CXCR4, albeit at variable levels, on leukaemic cells (Extended Data Fig. 1). We visualised T-ALL cells relative to osteoblasts (GFP or CFP positive in Col2.3-GFP/CFP reporter mice17,18), perivascular mesenchymal stem/progenitor cells (GFP positive in nestin-GFP reporter mice19) and vasculature (by injecting Cy5 dextran) in cohorts of recipient mice during disease progression and treatment (Fig. 1d).
By day 10 post-transplantation we could reproducibly observe single, sparse T-ALL cells in the BM at a frequency of 1-30 cells per calvarium (Fig. 1e, f). We measured the proximity of leukaemia cells to osteoblastic and nestin-GFP+ cells and vasculature. We used randomly positioned dots as a control for the specificity of observed associations, as these do not possess any inherent ability to localise to a particular BM stroma component (Fig. 1g). The distribution of T-ALL cells was equivalent to that of the random dots and the actual distances recorded inversely correlated with the abundance of each component (Fig. 1g-j). These results demonstrate that seeding T-ALL in the BM is stochastically distributed relative to osteoblasts, nestin-GFP+ cells and vasculature.
To determine whether T-ALL expansion was supported by specific constituents of the BM, we monitored the dynamics of single T-ALL cells (Fig. 2a) for 3 hours (Fig. 2b, c, Extended Data Fig. 2 and Supplementary Videos 2 and 3). This revealed that the vast majority of T-ALL cells were motile, in stark contrast with previous observations of transplanted haematopoietic stem cells in BM15, and that movement was rarely restricted to the proximity of any specific cell types or structures (Position 2 and 3 in Fig. 2b, c, and Supplementary Videos 2 and 3). Notably, the speed of any given cell over time was also heterogeneous, and thus no single migratory behaviour was associated with osteoblastic, nestin-GFP+ cells or vasculature (Fig. 2c, Extended Data Fig. 2 and Supplementary Videos 2 and 3). Tracking single T-ALL cells enabled us to measure the location of mitotic events, revealing the same stochastic distribution of dividing cells and suggesting that proximity to these stroma components is not key for T-ALL expansion (Fig. 2d, e, Extended Data Fig. 2, Supplementary Videos 2 and 3). Additionally, daughter cells migrated large distances following division, illustrating that clones and their progeny are not restricted to foci within the local microenvironment (Fig. 2c and Supplementary Video 2, position 3). These observations were consistent with tilescans performed at later stages of BM colonisation (day 12-18 post transplantation), where we detected pockets of high and low infiltration juxtaposed in all types of microenvironments (Extended Data Fig. 3 and and4).4). Interestingly, the motility patterns displayed by single, isolated cells were also consistently observed when tracking individual cells located in densely infiltrated areas (Supplementary Video 4). Combined, these analyses demonstrate that T-ALL seeding and colonisation of BM do not select for, or depend on specific BM stroma.
The question remained whether certain BM regions could create “hotspots” for chemo-resistance through provision of a protective environment. To address this issue, we adapted our imaging protocol to follow the same BM areas over multiple days to track leukaemia dynamics from complete BM infiltration and throughout therapy. We used the lock-and-key mechanism of the imaging window to precisely re-position mice on the microscope stage over multiple imaging sessions. Mice were tilescanned 18 days post T-ALL transplantation to confirm full BM infiltration. Dexamethasone was then administered daily (Fig. 3a), and immediately after the third therapy dose we observed a staggering reduction in disease burden20 (Fig. 3b-d and Extended Data Fig. 5a-b), while non-leukaemic, dexamethasone-treated control mice maintained robust BM and stroma cellularity (Extended Data Fig. 5d-e). Strikingly, surviving cells were scattered throughout the BM space and not preferentially associated with osteoblastic, nestin-GFP+ cells or vasculature compared to simulated data (Fig. 3b-f, Extended Data Fig 5a-c).
To test whether initial T-ALL loss was independent from specific stroma components, we increased the imaging temporal resolution to include the first day of treatment. The distribution of T-ALL cells was maintained as disease gradually succumbed to therapy (Extended Data Fig. 6). In contrast to predictions based on previous publications3,9,10, time-lapse imaging immediately after administration of the third dose of dexamethasone demonstrated that single surviving cells were highly migratory and did not maintain long-lasting associations with osteoblastic (Fig 3g, h and Supplementary Video 5) or nestin-GFP+ cells (Supplementary Video 6). Furthermore, surviving cells travelled at significantly faster speeds than early infiltrating cells (days 10-15) (Fig. 3m). Finally, residual T-ALL cells were still capable of undergoing division (Fig. 3g, Supplementary Videos 5 and 6) at times when other cells within the same mouse were still undergoing death (Supplementary Video 6). This behaviour, coupled with the observation that mice maintained on dexamethasone for a 7-day period harbored an almost completely repopulated BM space (Extended Data Fig. 7a), suggests these cells were genuinely resistant to dexamethasone.
Collectively, these data oppose the prevailing hypothesis that therapy-resistant leukaemic cells depend on a particular microenvironment for survival. To investigate this proposed paradigm, we compared the gene expression profile of T-ALL cells purified at full infiltration to those isolated from mice 7-10 days after initiation of dexamethasone treatment, when surviving cells have re-colonised the BM (Extended Data Fig. 7a). Gene expression profiles of all T-ALL samples were more heterogeneous compared to control T cells (CD4+ and CD8+), T cell progenitor (CD4+CD8+ thymocytes) and whole BM populations as expected based on inter-clone variation in leukaemia cells21. The transcriptome of resistant cells overlapped with that of T-ALL cells pre-treatment (Extended Data Fig. 7c). Indeed, only 79 genes were differentially expressed in the post-treatment group and, consistent with our intravital imaging data, none of the differentially expressed genes were related to known cell-niche interaction candidates (Supplementary Table 1 and Extended Data Fig. 7b). Together, our imaging and gene expression data suggest that dexamethasone treatment does not select for a subpopulation of T-ALL cells that have been directed to a specific niche.
To investigate the significance of these data in a human context, we assessed the migratory behaviour of xenotransplanted primary human T-ALL (Supplementary Table 2) in NOD/SCID/γ mice. Following dexamethasone treatment, even human wild-type Notch receptor T-ALL cells exhibited migratory behaviour equivalent to murine cells (Extended Data Fig. 8a-d and Supplementary Video 7). We noted an even distribution of quiescent cells throughout the BM as measured by Ki-67 staining (Extended Data Fig. 8e-f). To test whether the migratory behaviour was a specific trait induced by dexamethasone, we treated murine T-ALL burdened animals with vincristine. The T-ALL response followed similar kinetics to that of dexamethasone-treated animals. Enhanced migration of resistant cells and cell division events were evident immediately after the third dose of vincristine (Figure 3i, j, m and Supplementary Video 8). This behaviour was even more pronounced when we combined dexamethasone, vincristine and L-asparaginase (Fig. 3k-m, Extended Data Fig. 9 and Supplementary video 9). In this context, we observed the highest migration speed of all therapies tested, despite over 50% of surviving cells being in G0 (Figure 3n, o), demonstrating quiescence is not a feature of niche-restricted immotile cells following chemotherapy. These findings were universal in all therapies tested in our studies, suggesting migration and lack of long-lasting interactions with the surrounding microenvironment are conserved features of chemo-resistance in T-ALL.
To assay whether T-ALL may affect BM structures, we performed time-lapse imaging in heavily disease-burdened, untreated mice. After day 18 we observed striking remodelling of osteoblasts (Fig. 4a, b). A 9-hour period of time-lapse imaging after full infiltration (day 19 onwards) revealed that osteoblastic cells underwent dramatic shrinking and blebbing (Fig. 4a and Supplementary Video 10), in stark contrast to early stages of disease (Supplementary Video 1) and to control Col2.3-GFP mice reconstituted with red fluorescent healthy BM (Fig. 4a, b, Extended Data Fig. 10a, b and Supplementary Video 10). TUNEL and cleaved caspase-3 histological stainings indicated osteoblasts were undergoing apoptosis (Fig. 4c). The loss of osteoblasts was so dramatic that virtually no GFP+ osteoblasts remained apparent by days 22-25 post transplantation (Fig. 4b, d). Similar results were also found for osterix+ osteo-progenitors (Fig. 4f), however, nestin-GFP+ cells were maintained (Fig. 4e and Extended Data Fig. 10c, d), and blood vessels could still be visualised (Extended Data Fig. 10e). Importantly, these findings were consistent in analyses performed using human T-ALL samples (Supplementary Table 2). We observed a significant reduction in osterix+ osteo-progenitors in NOD/SCID/γ recipient mice xenotransplanted with human T-ALL samples (Fig. 4f). Additionally, histological analysis of BM trephine biopsies from T-ALL patients with greater than 75% infiltration demonstrated an almost complete loss of osteoblasts (Fig. 4g).
In summary, we demonstrate T-ALL infiltrates the BM and survives chemotherapy independently of stable interactions with specific microenvironments. However, T-ALL has a profound effect on osteoblastic cells. Our results suggest that to avoid the development of chemo-resistance, novel therapeutic interventions may not need to target specific BM stroma components, but rather the ability of T-ALL cells to interact with and migrate through the BM. Our studies also reveal that T-ALL has the intrinsic potential to remodel the BM microenvironment via apoptosis throughout the osteolineage. As these cells are associated with haematopoietic fitness14,22,23, including in the context of leukaemia1,2,24, the remodelling we uncovered may contribute to the loss of healthy haematopoiesis observed in leukaemia patients1,2. Collectively, our observations suggest that a shift in therapeutic design may be advisable, such that any intervention targeting osteoblastic cells should focus on promoting survival of this lineage, rather than seeking to modulate a direct influence of the BM on the cancer itself. Therefore, a better focus for novel anti-cancer therapeutics may be on disrupting the ability of T-ALL cells to interact transiently with multiple components of the BM microenvironment.
All animal experimentation in this study was approved and performed according to the standards of the animal ethics committee at Imperial College London, UK and to UK home office regulations (ASPA 1986). C57Bl/6 mice were purchased from Harlan UK Ltd; Col2.3-GFP, Col2.3-CFP18, Nestin-GFP and mTmG26 mice were bred and housed at Imperial College London, UK. For imaging experiments, female Col2.3-GFP and Nestin-GFP mice >8 weeks old were used. Osterix-CreGFP mice were provided by A. McMahon and backcrossed over 8 generations into NSG mice and maintained at the Francis Crick Institute, Cancer Research UK27. Equal proportions of male and female Osterix-CreGFP mice aged 11-14 weeks were used.
T-ALL was generated as previously described in28. Briefly, timed matings were established between C57/Bl6 mice and embryos harvested at day E14.5. Single cell suspensions were prepared from whole foetal livers isolated from the embryos. Suspensions were cultured in IL-3, IL-6, and stem cell factor conditioned media with 20% FCS for 3 days. Lin-xE cells were transfected by calcium phosphate with MigR1 plasmids containing either DsRed only or DsRed with NotchICNΔRamΔP as originally described in29. We also used GFP tagged plasmids when required. Supernatants containing recombinant retrovirus were removed and spun by centrifugation onto non-tissue culture treated plates coated with 15ug/ml retronectin (Takara Clontech, CA). Foetal liver cells were cultured in the presence of virus for 3 days and transduction assessed by flow cytometry. Primary lethally irradiated recipient mice (two doses of 5.5Gy administered greater than three hours apart) were transplanted with 1x106 DsRed+ve foetal liver cells by intravenous injection into the tail vein. Recipient mice were maintained on baytril treated water to prevent infection for >6 weeks post transplantation. Cohorts of reconstituted mice were the result of 3 independent foetal liver isolations and 3 independent transfections. Transformation of Notch-transduced non-malignant cells is highly heterogeneous in vivo, with onset of primary disease ranging from 6-25 weeks29 (Extended Data Fig. 1). >4 weeks post reconstitution, peripheral blood was isolated from mice, red blood cells were lysed, and successful reconstitution determined by presence of DsRed+ cells. Mice reconstituted with NotchICNΔRamΔP transduced foetal livers were monitored daily for signs of leukaemia onset or other signs of ill health. Mice were euthanized when any one, or combination of the following signs were observed: hunched posture, laboured breathing, weight loss, enlarged lymph nodes and/or spleen, peripheral white blood cell cellularity of 13 x109/L or greater. No experiment exceeded the tumour burden approved by the Home Office and Imperial College ethics committee. Peripheral lymphoid organs were analysed by flow cytometry for DsRed or GFP, CXCR4, CD3, CD4 and CD8 expression. All FACS data was collected on a Fortessa flow cytometer (BD Biosciences, CA). Secondary recipients were sub-lethally irradiated (two doses of 3Gy administered greater than three hours apart) and injected with 10,000 thawed, Ficoll purified T-ALL blasts and monitored as described above. In selected cases 10,000 secondary T-ALL cells were transplanted into tertiary recipients. For therapy experiments, mice were injected I.V daily with 15mg/kg dexamethasone sodium phosphate30,31 (Sellekchem, MA) alone, 0.15mg/kg vincristine sulfate salt (Sigma) alone or with a combination of 15mg/kg dexamethasone, 0.15mg/kg vincristine and 1,000 IU/kg L-asparaginase (medac; obtained from the Imperial College Healthcare NHS Trust Pharmacy).
Reconstitution with MigR1 DsRed transduced cells yields <50% chimerism. For this reason, analysis of healthy BM cells by microscopy is inaccurate. Therefore, to obtain >95% chimerism of healthy, red fluorescent BM to be used for imaging control experiments, whole BM mononuclear cells were isolated from femurs, hips and tibia of mTmG donor mice, suspended in phosphate balanced salt solution and administered intravenously to recipient mice at a dose of 2x106 cells/mouse. Recipient Col2.3-GFP or C57Bl/6 mice had been lethally irradiated (two doses of 5.5Gy irradiation greater than 3 hours apart) immediately prior to the transplant, and were maintained on baytril treated water to prevent infection >6 weeks post transplantation.
Intravital microscopy was performed using a Leica SP5 and a Zeiss LSM 780 upright confocal microscope with a motorised stage. The SP5 was fitted with the following lasers: Argon, 546, 633 and a tunable infrared multiphoton laser (Spectraphysics Mai Tai 690-1020). The Zeiss LSM 780 was fitted with the following lasers: Argon, 561, 633 and a tunable infrared multiphoton laser (Spectraphysics Mai Tai DeepSee 690-1040). Signal was visualised with a Leica HCX IRAPO L 25x water immersion lens (0.95 N.A) and a W Plan-Apochromat 20x DIC water immersion lens (1.0 N.A). Collagen bone second harmonic generation signal and GFP signal were generated through excitation at 840 nm and detected with external detectors. Internal detectors were used to collect DsRed and Cy5 signal (and on some occasions, GFP). Prior to surgery, mice were administered analgesia with buprenorphine (0.1mg/kg I.P). Anaesthesia was induced in mice with 4% isoflurane mixed with pure oxygen. This was gradually reduced to approximately 1% as anaesthesia stabilised. Surgery to attach the headpiece was then performed as described in16. Large 3 dimensional ‘tilescans’ of the entire BM cavity space were acquired by stitching adjacent, high-resolution Z-stack images using a surgically implanted imaging window that ensures steady positioning of mice on the microscope. The calvarium has been demonstrated to be equivalent to the long bones such as the femur with regards to haematopoietic stem cell frequency, function and localisation14,22, and is the only bone marrow compartment that allows longitudinal imaging through minimally invasive surgery16,32. Blood vessels were highlighted by I.V. injection of 50μl of 8μg/ml 500kD Cy5-Dextran (Nanocs, MA). Cy5 Dextran was re-injected every one-two hours to maintain blood vessel signal and cross reference for registration of blood vessel data in time-lapse analysis. For repeated imaging, protective intrasite gel (Smith & Nephew, UK) was applied to the imaging window to preserve the bone integrity and prevent scar formation. The window was bandaged, and mice were allowed to recover from anaesthesia. Due to the lock-and-key mechanism of the imaging window16, mice could then be re-anaesthetised and accurately repositioned on the microscope stage and the same BM areas re-imaged. After each imaging, analgesia was administered via oral buprenorphine in raspberry jelly at a dose of approximately 0.8mg/kg.
Microscopy data was processed using multiple platforms. Tilescans were stitched using Leica Application Systems (LAS, Leica Microsystems, Germany) and ZEN black (Zeiss, Germany) softwares. Raw data was visualised and processed using Fiji/Image J. Simulated data was prepared using FIJI macro to create, and overlay z-stack images on original tilescan data. Using the internal random number algorithm, spheres matching the size of T-ALL cells (11-15μm) were placed at random x,y,z co-ordinates. Simulated data FIJI macro is available on request. Automated cell segmentation, distance and volume measurements were performed in Definiens (Definiens Developer 64, Germany) using local heterogeneity segmentation25 to isolate osteoblast and Nestin cells as well as vasculature, and a combination of seed detection algorithm and morphological growing and shrinking operations to detect leukaemia cells. Definiens rulesets for these functions are available upon request. Distance measurements from this segmentation were performed as described in25. Cell tracking was performed using Imaris (Bitplane, Switzerland) and the FIJI plugin MTrackJ. For accuracy in cell tracking data, videos were registered when required before using 4D data protocols implemented in Fiji33. 3D data rendering and measurement of cell division distances were performed in Volocity (Perkin Elmer, MA) and Definiens (Definiens Developer 64, Germany).
T-ALL samples were harvested from bone marrow and FACS sorted based on fluorescent protein expression (DsRed or GFP) unless infiltration of bone marrow was complete. Control samples for microarray were prepared by FACS sorting for splenic CD4+ T cells, CD8+ T cells and CD4+CD8+ thymocytes from 8-14 week female C57Bl/6 mice. RNA was purified from samples using the Qiagen RNeasy mini kit (Netherlands) as per the manufactures instructions. Purified RNA was prepared for hybridisation using the Genechip® WT Plus reagent kit (Affymetrix, CA, USA) as per the manufactures instructions and hybridised with genechip® Mouse gene 2.0 ST array (Affymetrix, CA, USA) by the MRC Genomics facility (Imperial college London, UK). Analysis was performed using R version 3.1.1. Data was normalised and summarised to “core” level using the RMA method from the oligo package (version 1.30.0)34. Annotation was downloaded from the Affymetrix NetAffx Query website. Differential expression was determined using limma version 3.22.735. Genes with Benjamini-Hochberg-adjusted p-value < 0.05 and absolute log-fold-change > 1 were deemed significant. Heatmaps of gene expression were generated with pheatmap package version 1.0.2. Gene expression data are available on ArrayExpress (accession number E-MTAB-4889).
Primary human T-ALL samples were obtained from the Barts Hospital (London, UK) after informed consent via a protocol approved by the East London Research Ethics Committee and carried out in accordance with the principles of the Helsinki declaration (see Supplementary Table 2 for details), prior to treatment being administered to the patients. Primary cells from two distinct patients were immunophenotyped, and CD45+/CD7+/CD4-/low/CD8-/low cells sorted and infused I.V. in a non-conditioned Osterix-CreGFP/NOD/SCID/γ recipient mice. Primary xenograft transplantation was assessed via peripheral blood sampling and/or bone marrow aspiration. Bone marrow and spleen-derived primary xenografts were infused I.V. in non-conditioned NOD/SCID/γ secondary recipient mice for therapy experiments. Intravital imaging was performed as described above. Human T-ALL cells were labelled by injecting 10 μg of PE-conjugated human CD45 antibody (clone HI30, Biolegend) 15-30 minutes prior to the imaging session. For dexamethasone therapy experiments, mice were treated with daily injections of 15mg/kg I.V31. Number of human T-ALL cells in therapy experiments was quantified using reference beads as described in36.
Hips and tibias were harvested and post-fixed overnight in periodate-lysine-paraformaldehyde fixative, at 4ºC. Bones were then washed with 0.1M phosphate buffer, cryoprotected in sucrose (10-30% gradient), for 48h, frozen in optimal cutting temperature compound (TissueTek) and stored at -80ºC. Sections were cut in a Leica Cryostat, using the Cryojane tape transfer system (Leica microsystems) and stored at -80ºC. For staining, slides were re-hydrated in PBS, permeabilised in 0.1% Triton X-100, blocked in 5% goat serum and incubated with primary antibodies overnight, at 4 °C. After washing in PBS, slides were incubated with secondary antibodies, counter-stained with 4,6-diamidino-2-phenylindole (DAPI, Invitrogen), washed in 0.1% Triton X-100 and mounted using Prolong Diamond antifade (Invitrogen). The following antibodies were used: Alexa Fluor 647 mouse anti-Ki-67 (B56, BD Biosciences, 1:50), PE-conjugated human CD45 antibody (HI30, BD Biosciences, 1:100), rabbit anti-cleaved caspase-3 (Asp175, Cell Signaling, 1:100), goat anti-rabbit IgG Alexa Fluor 633 (Life Technologies, 1:400). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) labelling was performed to detect apoptotic cells, according to the manufacturer’s instructions (DeadEnd Colorimetric TUNEL System, Promega). Images were obtained using a Zeiss LSM 780 upright confocal/two-photon combined microscope and analysed using Fiji/ImageJ. Cell counting was performed manually using the FIJI plugin Cell Counter.
Bone marrow from human T-ALL xenotransplanted, untreated and treated mice was harvested and stained with DAPI (Invitrogen) and FITC mouse anti-Ki-67 set (BD Biosciences), according to the manufacturer’s instructions. Cells were analysed by FACS and absolute numbers were obtained using reference beads as described in36.
T-ALL engraftment and infiltration was confirmed via peripheral blood sampling and/or tibia puncture. Once mice presented with signs of ill health (as described earlier), mice were euthanized and bones digested with a DNAse I/Collagenase (Sigma) solution. The total number of Osx/GFP+ cells was assessed by flow cytometry analysis using counting beads (CountBright, Life Technologies).
Samples were obtained from patients after informed consent had been obtained, under full ethical approval by the Peter MacCallum Cancer Centre Human Research Ethics Committee. De-waxed human trephine biopsy sections (3μM) were stained with osteocalcin antibody (Abcam ab93876, Cambridge, UK), counterstained and mounted for viewing. All areas of each section were monitored for visible osteoblasts.
The sample size required for the experiments was estimated based of the results of preliminary data. Blinding or randomisation for animal experiments were not necessary due to the nature of the experiments. Statistical differences between the means of two data groups was determined by using two-tailed unpaired Student’s t test, and p values <0.05 were considered significant. Multiple group comparisons were performed using ANOVA with a Bonferroni correction, p values <0.05 were considered significant.
EH was supported by a fellowship from the European Haematology Association (EHA), and project grant from Bloodwise (12033). DD was supported by the GABBA PhD program (FCT fellowship SFRH/BD/52195/2013). LP was supported by an NHMRC Senior Research Fellowship. HQ is supported by Victorian Cancer Agency Early Career Seed Grant. DB, DP and KF were funded by Cancer Research UK and the Francis Crick Institute. CLC was funded by Bloodwise (12033), HFSP (RGP0051/2011), CRUK (C36195/A1183), BBSRC (BB/I004033/1) KKLF (KKL460) and ERC (337066). We are grateful to Dr. Martin Spitaler and Dr. Debbi Keller (Imperial College FILM facility) for support with microscopy. We also thank Savvas Piperelis, Edgar Ibarguen, Wendy Steel and Helen Goyal (Imperial College CBS facility) for logistical help; Andrew Ivan for support from the Genomics facility; Jane Srivastava and Catherine Simpson for support from the flow cytometry facility; Meaghan Wall (St. Vincent's Hospital) for her advice on the human cytogenetics; Delissa Ibanez (Bitplane) for access to software; Dr. H. Fleming (MIT), Prof. V. Greco (Yale University), Prof. RE Sinden (IC), Dr S. Mostowy (IC) and Dr. P. O’Donovan (Wellcome Trust) for critical feedback on the manuscript.
Author contributionsEH and CLC conceived the project. EH, CLC, OA, MS designed and refined the intravital imaging systems. RK designed the image analysis platform; RK, EH and DD performed all image analysis. SR designed the random data simulator. EH, DD and NR performed imaging experiments. NR maintained all animal lines. MN and MR designed and analysed microarray data. EH, MW and RJ performed experiments in Extended Data Fig. 1. DP, KF and DB designed, performed and analysed Osterix/GFP experiments. SH, DW and JG provided human T-ALL samples. LS and LP performed histological analysis of human BM trephines. HQ funded human biopsy studies. DP and DB designed and DP performed the human T-ALL xenograft experiments and analysed data. EH, DD and CLC analysed data and wrote the manuscript. Every author contributed revisions to the manuscript.
Competing financial interests
The authors have no competing financial interests regarding to the work in this manuscript.