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• W3197373195 abstract "•Some bone marrow stroma cells extensively phagocytose apoptotic cells•Disappearance of dead cells from cocultures due to phagocytosis confounds results•This needs to be considered in studies using relative viabilities in cocultures•Bone marrow stroma cell line NKTert could also phagocytose glass spheres Signals provided by the microenvironment can modify and circumvent pathway activities that are therapeutically targeted by drugs. Bone marrow stromal cell coculture models are frequently used to study the influence of the bone marrow niche on ex vivo drug response. Here, we show that mesenchymal stromal cells from selected donors and NKTert, a stromal cell line, which is commonly used for coculture studies with primary leukemia cells, extensively phagocytose apoptotic cells. This could lead to misinterpretation of results, especially if viability readouts of the target cells (e.g. leukemic cells) in such coculture models are based on the relative proportions of dead and alive cells. Future coculture studies which aim to investigate the impact of bone marrow stromal cells on drug response should take into account that stromal cells have the capacity to phagocytose apoptotic cells. Signals provided by the microenvironment can modify and circumvent pathway activities that are therapeutically targeted by drugs. Bone marrow stromal cell coculture models are frequently used to study the influence of the bone marrow niche on ex vivo drug response. Here, we show that mesenchymal stromal cells from selected donors and NKTert, a stromal cell line, which is commonly used for coculture studies with primary leukemia cells, extensively phagocytose apoptotic cells. This could lead to misinterpretation of results, especially if viability readouts of the target cells (e.g. leukemic cells) in such coculture models are based on the relative proportions of dead and alive cells. Future coculture studies which aim to investigate the impact of bone marrow stromal cells on drug response should take into account that stromal cells have the capacity to phagocytose apoptotic cells. Signals provided by the microenvironment protect leukemia cells from spontaneous apoptosis and modify pathway activities which are therapeutically targeted by drugs (Choi et al., 2016Choi M.Y. Kashyap M.K. Kumar D. The chronic lymphocytic leukemia microenvironment: beyond the B-cell receptor.Best Pract. Res. Clin. Haematol. 2016; 29: 40-53Crossref PubMed Scopus (17) Google Scholar; Ten Hacken and Burger, 2016Ten Hacken E. Burger J.A. Microenvironment interactions and B-cell receptor signaling in Chronic Lymphocytic Leukemia: implications for disease pathogenesis and treatment.Biochim. Biophys. Acta. 2016; 1863: 401-413Crossref PubMed Scopus (126) Google Scholar). In vitro coculture models of malignant cells and various bone marrow stromal cell types have been frequently used to understand tumor – microenvironment interactions and how these interactions interfere with the response to cancer drugs. Most commonly, cocultures of leukemia cells and the human bone marrow stromal cell lines NKTert and HS-5, or primary mesenchymal stromal cells (MSCs) are used as models of the human bone marrow niche. Several reports suggest that NKTert and other stromal cells protect leukemia cells from spontaneous and drug-induced apoptosis (Balakrishnan et al., 2015Balakrishnan K. Peluso M. Fu M. Rosin N.Y. Burger J.A. Wierda W.G. Keating M.J. Faia K. O’Brien S. Kutok J.L. Gandhi V. The phosphoinositide-3-kinase (PI3K)-delta and gamma inhibitor, IPI-145 (Duvelisib), overcomes signals from the PI3K/AKT/S6 pathway and promotes apoptosis in CLL.Leukemia. 2015; 29: 1811-1822Crossref PubMed Scopus (116) Google Scholar; Cheng et al., 2014Cheng S. Ma J. Guo A. Lu P. Leonard J.P. Coleman M. Liu M. Buggy J.J. Furman R.R. Wang Y.L. BTK inhibition targets in vivo CLL proliferation through its effects on B-cell receptor signaling activity.Leukemia. 2014; 28: 649-657Crossref PubMed Scopus (137) Google Scholar; Fiorcari et al., 2013Fiorcari S. Brown W.S. McIntyre B.W. Estrov Z. Maffei R. O’Brien S. Sivina M. Hoellenriegel J. Wierda W.G. Keating M.J. et al.The PI3-kinase delta inhibitor idelalisib (GS-1101) targets integrin-mediated adhesion of chronic lymphocytic leukemia (CLL) cell to endothelial and marrow stromal cells.PLoS ONE. 2013; 8: e83830Crossref PubMed Scopus (71) Google Scholar; Kurtova et al., 2009Kurtova A.V. Balakrishnan K. Chen R. Ding W. Schnabl S. Quiroga M.P. Sivina M. Wierda W.G. Estrov Z. Keating M.J. et al.Diverse marrow stromal cells protect CLL cells from spontaneous and drug-induced apoptosis: development of a reliable and reproducible system to assess stromal cell adhesion-mediated drug resistance.Blood. 2009; 114: 4441-4450Crossref PubMed Scopus (245) Google Scholar; Zhang et al., 2012Zhang W. Trachootham D. Liu J. Chen G. Pelicano H. Garcia-Prieto C. Lu W. Burger J.A. Croce C.M. Plunkett W. et al.Stromal control of cystine metabolism promotes cancer cell survival in chronic lymphocytic leukaemia.Nat. Cell Biol. 2012; 14: 276-286Crossref PubMed Scopus (213) Google Scholar). Here, we demonstrate that NKTert and primary MSCs from selected donors massively phagocytose apoptotic cells in vitro. The clearance of dead cells increases the relative proportion of alive cells in these cocultures, which represents a major source of bias if the viability readout is based on relative proportions of alive and dead cells. In contrast, other cocultures (with some MSCs or HS-5) did not show this behavior, suggesting them as suitable feeder cells for coculture drug response studies. Leukemic cells from four patients with chronic lymphocytic leukemia (CLL) were cultured either alone (monocultures) or in coculture with the NKTert stromal cell line. The monocultures and cocultures were treated with solvent control (DMSO), venetoclax, or fludarabine. After 72 h, the cultures were stained with the nuclear dye SiR-DNA, the viability dye calcein and the dead cell marker propidium iodide (PI). Based on these stainings, we quantified dead and alive CLL cells by flow cytometry and high content confocal microscopy. First, we calculated relative proportions of dead and alive cells. In accordance with the literature (Balakrishnan et al., 2015Balakrishnan K. Peluso M. Fu M. Rosin N.Y. Burger J.A. Wierda W.G. Keating M.J. Faia K. O’Brien S. Kutok J.L. Gandhi V. The phosphoinositide-3-kinase (PI3K)-delta and gamma inhibitor, IPI-145 (Duvelisib), overcomes signals from the PI3K/AKT/S6 pathway and promotes apoptosis in CLL.Leukemia. 2015; 29: 1811-1822Crossref PubMed Scopus (116) Google Scholar; Cheng et al., 2014Cheng S. Ma J. Guo A. Lu P. Leonard J.P. Coleman M. Liu M. Buggy J.J. Furman R.R. Wang Y.L. BTK inhibition targets in vivo CLL proliferation through its effects on B-cell receptor signaling activity.Leukemia. 2014; 28: 649-657Crossref PubMed Scopus (137) Google Scholar; Fiorcari et al., 2013Fiorcari S. Brown W.S. McIntyre B.W. Estrov Z. Maffei R. O’Brien S. Sivina M. Hoellenriegel J. Wierda W.G. Keating M.J. et al.The PI3-kinase delta inhibitor idelalisib (GS-1101) targets integrin-mediated adhesion of chronic lymphocytic leukemia (CLL) cell to endothelial and marrow stromal cells.PLoS ONE. 2013; 8: e83830Crossref PubMed Scopus (71) Google Scholar; Kurtova et al., 2009Kurtova A.V. Balakrishnan K. Chen R. Ding W. Schnabl S. Quiroga M.P. Sivina M. Wierda W.G. Estrov Z. Keating M.J. et al.Diverse marrow stromal cells protect CLL cells from spontaneous and drug-induced apoptosis: development of a reliable and reproducible system to assess stromal cell adhesion-mediated drug resistance.Blood. 2009; 114: 4441-4450Crossref PubMed Scopus (245) Google Scholar; Zhang et al., 2012Zhang W. Trachootham D. Liu J. Chen G. Pelicano H. Garcia-Prieto C. Lu W. Burger J.A. Croce C.M. Plunkett W. et al.Stromal control of cystine metabolism promotes cancer cell survival in chronic lymphocytic leukaemia.Nat. Cell Biol. 2012; 14: 276-286Crossref PubMed Scopus (213) Google Scholar), we could confirm higher proportions of alive CLL cells in cocultures with NKTert stromal cells than in suspension monocultures of CLL cells only. This was true for all untreated and drug treated conditions and could be interpreted as stromal-cell-mediated protection from spontaneous and drug-induced apoptosis (Figure 1A). We further compared absolute CLL cell counts as determined by microscopy and flow cytometry. Surprisingly, we observed much lower absolute CLL cell counts in NKTert cocultures than in CLL monocultures (Figure 1B). This decrease was due to the disappearance of dead cells (Figure 1C, left). Absolute counts of alive cells in the cultures did not differ between monocultures and cocultures with NKTert (Figure 1C, right). To better understand this finding, we labeled leukemia cells from four other patients with CLL with CellTracker Green, pretreated them with venetoclax and cocultured them with CellTracker Blue labeled NKTert cells (Figure 1D). Apoptotic leukemia cells disappeared after 16 h in coculture with NKTert cells, while apoptotic cells were still present in monocultures. This finding could be confirmed in an additional experiment comprising six more patients with CLL using microscopy (Figure S1) or flow cytometry and annexin/PI staining (Figure S2). We further aimed to understand whether other stromal cells behave in the same way and cocultured venetoclax pretreated CLL cells with the cell line HS-5. Cell counts of CLL cells were comparable between monocultures of CLL cells and cocultures with HS-5 stromal cells. These experiments indicated that apoptotic leukemia cells disappeared in NKTert, but not in HS-5 cocultures, which increased the relative proportion of alive leukemia cells (Figure 1A) and could falsely be interpreted as protection of CLL cells by NKTert from spontaneous or drug-induced apoptosis. Next, we aimed to identify why apoptotic cells disappeared in NKTert cell line cocultures. Especially in the venetoclax-treated cocultures, many CLL cells seemed to be located inside of NKTert, based on bright field microscopy (Figures 2A and S3). Confocal microscopy could confirm the localization of CellTracker-Green-labeled CLL inside of CellTracker-Blue-labeled NKTert (Video S1). Further staining with the lysosomal dye NIR revealed large lysosomal bodies inside NKTert cells (Figure 2B), which had the size and shape of CLL cells. Lysosomal bodies often occurred in regions also positive for CellTracker Green and PI staining and were surrounded by, but did not include, CellTracker Blue staining (Figure 2C and Video S1). Time lapse confocal microscopy revealed that uptake of CellTracker Green-labeled CLL cells by NKTert cells and concurrent formation of these phagosomes inside NKTert cells occurred in less than 10 min (Figure S4). We quantified the amount of phagosomes in three independent experiments (Figure 2D). A high number of phagosomes were present in treated NKTert cocultures, whereas large lysosomal bodies were absent in CLL monocultures, NKTert, or HS-5 stroma monocultures or cocultures of CLL and HS-5 cells. The formation of phagosomes could not be prevented by the addition of the caspase inhibitor zVAD to venetoclax-treated cultures (Figure S5). We concluded that NKTert cells, but not HS-5, phagocytose dying CLL cells and, thereby, clear those cells from the cocultures. We wondered whether phagocytosis by NKTert was specific to CLL cells. To this end, the mantle cell lymphoma cell line HBL-2, the acute myeloid leukemia cell line OCI-AML 2, the carcinoma cell line HELA, and the benign epithelial cell line HEK-293T were exposed to 10 μM doxorubicine, to induce apoptosis. Cells were then cocultured with NKTert cells. NKTert phagocytosed apoptotic cells of all tested cell lines, regardless of their origin (Figures 3A and S6). In conclusion, phagocytic activity by NKTert cells is not limited to CLL cells, but is likely cell type independent. https://www.cell.com/cms/asset/ca3bf795-b1f1-49e5-a1ba-741b82487798/mmc3.mp4Loading ... Download .mp4 (1.88 MB) Help with .mp4 files Video S1. NKTert cell containing phagocytosed CLL cells, related to Figures 2A and 2C3D view of confocal microscopy (63× objective) of co-culture of CellTracker Blue labeled NKTert and CellTracker Green labeled CLL cells treated with venetoclax. Green = CellTracker Green, blue = CellTracker Blue, yellow = propidium iodide, red = lysosomal dye NIR, black and white = bright field image. https://www.cell.com/cms/asset/8f21e2f3-40dc-48a5-8f72-fafed275840c/mmc4.mp4Loading ... Download .mp4 (8.39 MB) Help with .mp4 files Video S2. MSC cell containing phagocytosed CLL cells, related to Figure 3B3D view of confocal microscopy (63× objective) of co-culture of CellTracker Blue labeled MSC cell from donor MSC1 and CellTracker Green labeled CLL cells treated with venetoclax. Green = CellTracker Green, blue = CellTracker Blue, yellow = propidium iodide, red = lysosomal dye NIR. To further evaluate whether the ability to phagocytose apoptotic cells is restricted to the cell line NKTert or whether it also occurs in primary MSCs, which are a more physiological model of the bone marrow niche than cell lines, we cultured venetoclax treated and CellTracker-Green-labeled primary CLL cells with CellTracker-Blue-labeled primary MSCs from four different healthy donors. Phagocytosed CLL cells were observed in MSCs from two out of four donors (Figure S7). Phagocytosis was especially high in MSC1 culture (Figure 3B, Video S2), while MSC2 only exhibited a low amount of phagocytosis (Figure S7). This shows that not only NKTert but also some but not all primary MSCs are able to phagocytose apoptotic cells. Due to the massive phagocytic activity exhibited by NKTert, we assessed whether or not the cells are of macrophagic origin. By performing staining against CD45 followed by flow cytometry, we could show that NKTert are negative for this pan-hematopoietic cell marker and, based on this, are not of monocytic lineage or conventional macrophage differentiation (Figure S8). This confirmed the findings by Kawano et al., 2003Kawano Y. Kobune M. Yamaguchi M. Nakamura K. Ito Y. Sasaki K. Takahashi S. Nakamura T. Chiba H. Sato T. et al.Ex vivo expansion of human umbilical cord hematopoietic progenitor cells using a coculture system with human telomerase catalytic subunit (hTERT)-transfected human stromal cells.Blood. 2003; 101: 532-540Crossref PubMed Scopus (131) Google Scholar. Therefore, the extensive phagocytosis by NKTert cells cannot be explained by a macrophagic origin of these cells. To further characterize NKTert and HS-5 cells, we performed additional flow cytometry staining with a panel of nine markers and a viability dye (Figure 4A). As expected, HS-5 and NKTert were positive for common MSC markers CD44, CD73, CD90, CD271, CD26, podoplanin, CD31, and CD105 but negative for the endothelial and pericyte marker CD146, thus confirming a mesenchymal origin of NKTert and HS-5 (Li et al., 2016Li H. Ghazanfari R. Zacharaki D. Lim H.C. Scheding S. Isolation and characterization of primary bone marrow mesenchymal stromal cells.Ann. N. Y. Acad. Sci. 2016; 1370: 109-118Crossref PubMed Scopus (68) Google Scholar; Lv et al., 2014Lv F.J. Tuan R.S. Cheung K.M. Leung V.Y. Concise review: the surface markers and identity of human mesenchymal stem cells..Stem Cells. 2014; 32: 1408-1419Crossref PubMed Scopus (562) Google Scholar). Interestingly, NKTert cells displayed a bimodal expression pattern of some markers, including CD90, podoplanin, and CD26, suggesting phenotypic and functional heterogeneity of NKTert. CD90, CD26, and podoplanin have been shown to facilitate fibroblast activation, migration, and extracellular matrix remodeling (Ghersi et al., 2002Ghersi G. Dong H. Goldstein L.A. Yeh Y. Hakkinen L. Larjava H.S. Chen W.T. Regulation of fibroblast migration on collagenous matrix by a cell surface peptidase complex.J. Biol. Chem. 2002; 277: 29231-29241Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar; Lee et al., 2020Lee S.Y. Wu S.T. Liang Y.J. Su M.J. Huang C.W. Jao Y.H. Ku H.C. Soluble dipeptidyl peptidase-4 induces fibroblast activation through proteinase-activated receptor-2.Front. Pharmacol. 2020; 11: 552818Crossref PubMed Scopus (2) Google Scholar; Martín-Villar et al., 2010Martín-Villar E. Fernández-Muñoz B. Parsons M. Yurrita M.M. Megías D. Pérez-Gómez E. Jones G.E. Quintanilla M. Podoplanin associates with CD44 to promote directional cell migration.Mol. Biol. Cell. 2010; 21: 4387-4399Crossref PubMed Scopus (89) Google Scholar; Rege et al., 2006Rege T.A. Pallero M.A. Gomez C. Grenett H.E. Murphy-Ullrich J.E. Hagood J.S. Thy-1, via its GPI anchor, modulates Src family kinase and focal adhesion kinase phosphorylation and subcellular localization, and fibroblast migration, in response to thrombospondin-1/hep I.Exp. Cell Res. 2006; 312: 3752-3767Crossref PubMed Scopus (33) Google Scholar; Suchanski et al., 2017Suchanski J. Tejchman A. Zacharski M. Piotrowska A. Grzegrzolka J. Chodaczek G. Nowinska K. Rys J. Dziegiel P. Kieda C. Ugorski M. Podoplanin increases the migration of human fibroblasts and affects the endothelial cell network formation: A possible role for cancer-associated fibroblasts in breast cancer progression.PLOS One. 2017; 12: e0184970Crossref PubMed Scopus (20) Google Scholar). Since only one out of the two tested human bone marrow stromal cell lines and two out of the four tested MSCs phagocytosed apoptotic cells, we were interested in molecular differences associated with this phagocytic activity. We performed quantitative proteomics of HS-5 and NKTert cells (Data S1). Pathway enrichment analysis on protein abundances revealed that proteins involved in focal adhesion and interaction with the extracellular matrix were significantly upregulated in NKTert cells, which is in accordance with the high abundance of CD90, CD26, and podoplanin in a subpopulation of NKTert. Additionally, the lysosomal pathway was most significantly enriched within differential proteins. Most proteins in this pathway were upregulated in NKTert in comparison to HS-5 (Figure 4B). We observed that blocking the fusion of lysosomes with phagosomes by treatment with chloroquine did not inhibit the uptake of apoptotic cells but prevented acidification of phagosomes and digestion of the content (Figure S9). This could indicate that upregulation of lysosomal proteins may contribute to the phagocytic activity of NKTert, by ensuring the efficient digestion of ingested cells. Finally, we aimed to assess whether phagocytosis by NKTert cells was mainly triggered by eat-me signals on dead cells or rather prevented by don't-eat-me signals on alive cells. For this we added glass spheres with a particle size of 9–13 μM to CellTracker Blue stained NKTert cultures and assessed the uptake of the spheres after 16 h of culturing. As shown in Figure 4C, NKTert cells ingested large amounts of glass spheres. All analyzed cells had taken up spheres, which indicates that the different populations observed within NKTert in flow cytometry do not possess different phagocytic activities. As described for macrophages (Gilberti et al., 2008Gilberti R.M. Joshi G.N. Knecht D.A. The phagocytosis of crystalline silica particles by macrophages.Am. J. Respir. Cell Mol. Biol. 2008; 39: 619-627Crossref PubMed Scopus (53) Google Scholar), this suggests that the differences between phagocytosis of alive and dead cells by NKTert arise due to the presence of don't-eat-me signals or specific surface charges on the membrane of the target cells. In conclusion, we found that NKTert and selected primary MSCs massively phagocytose apoptotic cells in-vitro. Although it has been reported that MSCs are capable of phagocytosis (Dogusan et al., 2004Dogusan Z. Montecino-Rodriguez E. Dorshkind K. Macrophages and stromal cells phagocytose apoptotic bone marrow-derived B lineage cells.J. Immunol. 2004; 172: 4717-4723Crossref PubMed Scopus (24) Google Scholar), the extent and consequences for in vitro assays have not been anticipated. Phagocytosis leads to the removal of dead cells from the cocultures causing an increased percentage of alive cells, which might be falsely interpreted as a protective effect mediated by stromal cells. This bias might affect flow cytometry assays which rely on the measurement of the relative proportion of alive and dead cells without assessing total cell counts in an in vitro culture model. Together this suggests that results from coculture experiments with NKTert or selected MSCs could be misinterpreted if not corrected for this potential bias triggered by the phagocytic activity of these cell types. There is no doubt that selected bone marrow stromal cells and cell lines are able to protect leukemia cells from spontaneous and drug-induced apoptosis (Lagneaux et al., 1998Lagneaux L. Delforge A. Bron D. De Bruyn C. Stryckmans P. Chronic lymphocytic leukemic B cells but not normal B cells are rescued from apoptosis by contact with normal bone marrow stromal cells.Blood. 1998; 91: 2387-2396Crossref PubMed Google Scholar). For some, but not all stromal cells, this effect might be confounded by their phagocytic activity. The cell line HS-5 for example did not phagocytose apoptotic cells and might therefore be a suitable coculture model system. For future studies, phagocytosis by stromal cells needs to be tested and taken into account, especially as the heterogeneity between cell lines or donors seems to be high. Even though first approaches into this direction have been taken (Baccin et al., 2020Baccin C. Al-Sabah J. Velten L. Helbling P.M. Grünschläger F. Hernández-Malmierca P. Nombela-Arrieta C. Steinmetz L.M. Trumpp A. Haas S. Combined single-cell and spatial transcriptomics reveal the molecular, cellular and spatial bone marrow niche organization.Nat. Cell Biol. 2020; 22: 38-48Crossref PubMed Scopus (150) Google Scholar; Baryawno et al., 2019Baryawno N. Przybylski D. Kowalczyk M.S. Kfoury Y. Severe N. Gustafsson K. Kokkaliaris K.D. Mercier F. Tabaka M. Hofree M. et al.A cellular taxonomy of the bone marrow stroma in homeostasis and leukemia.Cell. 2019; 177: 1915-1932.e16Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar), stromal cells are still not sufficiently characterized. Only this will ensure accurate determination of the influence of the microenvironment and, thus, the development of effective treatment strategies. This study demonstrates how phagocytic activity of some bone marrow stromal cells can confound results produced in cocultures with these cells. The presented data do not exclude the fact and do not provide to which extent bone marrow stroma cells possess the ability to protect leukemic cells from apoptosis. This needs to be thoroughly investigated in larger and more comprehensive studies. Additionally, we could show that NKTert cells also phagocytose glass spheres and that, therefore, phagocytosis is most likely dependent on don't-eat-me signals or specific surface charges on the membrane of the target cells. However, the exact mechanism or the factors driving the difference in phagocytic activity between stromal cells remains to be uncovered. Tabled 1REAGENT or RESOURCESOURCEIDENTIFIERAntibodiesCD146-BV421 (P1H12)BiolegendCat# 361003; RRID: AB_2562966CD31-BV650 (M89D3)BD BiosciencesCat# 744465; RRID: AB_2742254CD105-FITC (43A3)BiolegendCat# 323203; RRID: AB_755955CD90-PerCP/Cy5.5 (5E10)BD BiosciencesCat# 561557; RRID: AB_10712762CD44-PE (IM7)BiolegendCat# 103023; RRID: AB_493686podoplanin-PE/Dazzle (NC-08)BiolegendCat# 337027; RRID: AB_2750287CD271-PE/Vio770 (ME20.4-1.H4)Miltenyi BiotecCat# 130113984; RRID: AB_2733219CD26-APC (M-A261)BD BiosciencesCat# 563670; RRID: AB_2738363CD73-APC/Cy7 (AD2)BiolegendCat# 344021; RRID: AB_2566755Chemicals, peptides, and recombinant proteinsHoechst 33342InvitrogenCat# 62249CellTracker BlueInvitrogenCat# C2111CalceinInvitrogenCat# C1430CellTracker GreenInvitrogenCat# C7025SiR-DNA kitSpirochromeLysosomal Staining Reagent - NIR - CytopainterAbcamCat# ab176824Experimental models: Cell linesHS-5 cell lineKind gift by Martina Seiffert, DKFZRRID: CVCL_3720NKTert cell lineRIKEN BRCRRID: CVCL_4667Deposited dataProteomics data of HS-5 and NKTertProteomeXchange Consortium via the PRIDE partner repositoryPRIDE: PXD027945Software and algorithmsDeposited codehttps://github.com/DietrichLab/Phagocytosis Open table in a new tab This study did not generate new unique reagents. Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Sascha Dietrich ( [email protected] ). HS-5 cells were a kind gift by Martina Seiffert. NKTert cells were obtained from RIKEN BRC. Cell lines were maintained in RPMI supplemented with 10% FBS, 1% Penicillin/Streptomycin and 1% Glutamine at 37°C and 5% CO2 in a humidified atmosphere. Primary MSCs were maintained in MSCGM Mesenchymal Stem Cell Growth Medium Bulletkit medium (Lonza). Cell lines were tested for mycoplasma before all experiments using a PCR based testing procedure. Authentication of HS-5 and NKTert cells was performed (Multiplexion). HS-5 cells could be successfully authenticated. For NKTert no reference sequence was available in the database, however, the NKTert DNA was identified as unique sequence, did not match to any other cell line and, therefore, cross contamination could be excluded. In the study the following dyes for microscopy were used at the indicated concentration. The indicated wave lengths were used for fluorophore excitation: Hoechst 33342 (4 μg/ml, Invitrogen, 405 nm), CellTracker Blue (10 μM, Invitrogen, 405 nm), Calcein (1 μM, Invitrogen, 488 nm), CellTracker Green (10 μM, Invitrogen, 488 nm), propidium iodide (PI; 5 μg/ml, Sigma-Aldrich, 561 nm), SiR-DNA (1 μM, Spirochrome, 640 nm), lysosomal dye NIR (1 μl/ml, Abcam, 640 nm). These dyes were used in the following combinations: Hoechst, Calcein, PI and lysosomal dye NIR, or Calcein, PI and SiR-DNA or CellTracker Blue, CellTracker Green, PI and lysosomal dye NIR. With the used setup no crosstalk between the channels was seen, with the exception of a slight spillover between calcein and PI and a slight spillover of the signal from the lysosomal dye into the channel for PI. This section describes the experiment shown in Figures 1A–1C. We cultured cancer cells from four different patients with CLL (2 × 105/well) either alone or in coculture with NKTert (1 × 104/well) in 96-well glass bottom microscopy plates (zell-kontakt GmbH). Stromal cells were seeded 24 hours before the addition of leukemia cells. The samples were treated with solvent control (DMSO), 63 nM venetoclax or 10 μM fludarabine. After 72 hours the cultures were stained with the nuclear dye SiR-DNA, the viability dye Calcein and the dead cell marker PI. Images were taken with the confocal LSM710 microscope (Zeiss) equipped with climate control (37°C, 5% CO2) using a 20× objective lens. Z-stack images were acquired in triplicate wells, and within one well 4 adjacent fields were imaged. Directly after acquisition of the confocal microscopy pictures, lymphocytes previously labeled with SiR-DNA, Calcein and PI were pipetted from each cultural condition to a 96-well round bottom plate (Greiner) for further analysis with an IQue Screener (Intellicyt). Calcein and PI signals were recorded. The following gating strategy was pursued: Exclusion of potentially remaining stromal cells, by setting of a lymphocyte gate and exclusion of doublets. The percentage of alive cells was determined by gating on Calcein positive and PI negative cells. This section describes the experiment shown in Figures 1D, 1E, S1, S5, and S9. We labeled leukemia cells from four or six other patients with CellTracker Green and cocultured them (2 × 105 cells/well) with CellTracker Blue labeled NKTert (1 × 104 cells/well) or HS-5 (2 × 104 cells/well) stromal cells. CLL cells were pretreated with solvent control or 63 nM venetoclax or 63 nM venetoclax and 10 μM Z-VAD-FMK (Sigma-Aldrich) for 24 hours. For chloroquine treatment, NKTert cells were pretreated with 20 μM hydroxychloroquine for 24 hours. Co-culturing was performed for 16 hours. Cultures were additionally stained with lysosomal dye NIR and PI before imaging. The samples were imaged on an Opera Phenix microscope (Perkin Elmer) in confocal mode. For clearer visualization, the signal from the lysosomal dye is not shown in Figure 1D, while CellTracker Green signal and PI staining are not shown Figure 1E. The images shown are representative of all experiments. This section describes the experiment shown in Figure S2. We labeled leukemia cells from six patients with CellTracker Green and cocultured them (2 × 105 cells/well) with CellTracker Blue labeled NKTert (1 × 104 cells/well) stromal cells. CLL cells were treated with solvent control or 63 nM venetoclax. After co-culturing for three days samples were harvested by vigorous pipetting. APC Annexin V (RUO; BD Biosciences)/PI staining was performed according to the manufacturer's recommendations. CountBright™ Absolute Counting Beads (ThermoFisher Scientific) were added before measurement. Acquisition was done on a FACSSymphony (BD Biosciences). Compensation, gating on CLL cells, singlets, CellTracker Green positive and CellTracker Blue negative cells was performed before plotting of Annexin against PI intensity. Cell counts were normalized to numbers of counting beads. This section describes the experiment shown in Figure 1F. The amount of phagosomes was quantified in three independent experiments comprising in total three different CLL patient sampl" @default.
• W3197373195 created "2021-09-13" @default.
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• W3197373195 date "2021-09-01" @default.
• W3197373195 modified "2023-10-18" @default.
• W3197373195 title "Phagocytosis by stroma confounds co-culture studies" @default.
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