The authors would like to acknowledge the "Ligue Nationale contre le Cancer" (Comit&#233; du Septentrion), the SIRIC ONCOLille (Grant INCa-DGOS-INSERM 6041) and the Institut pour la Recherche sur le Cancer de Lille (IRCL) for supporting this work. They would like to thank Delphine Taillieu and the animal facility staff for housing the mice and maintaining their welfare. We also thank Rapha&#235;lle Caillerez and Nathalie Jouy for their respective help in microscopy and flow cytometry.

<ol>
	<li>Forthun, R. B., Hinrichs, C., Dowling, T. H., Bruserud, &. #. 2. 1. 6. ;., Selheim, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+past,+present+and+future+subclassification+of+patients+with+acute+myeloid+leukemia.">The past, present and future subclassification of patients with acute myeloid leukemia.</a> Curr. Pharm. Biotechnol. 17 (1), 6-19 (2016).</li><li>Goldie, H., Butler, C. H., Anderson, M. M., Maxwell, M. C., Hahn, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+characteristics+of+free+C1498+(granulocytic+leukemia)+tumor+cells+in+the+peritoneal+fluid+and+the+blood+of+C57+mice.">Growth characteristics of free C1498 (granulocytic leukemia) tumor cells in the peritoneal fluid and the blood of C57 mice.</a> Cancer Res. 13 (2), 125-129 (1953).</li><li>Tanaka, K. K., Roberts, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+studies+of+E.L.4+lymphoma+and+C+Leukemia+in+susceptible+(C57BL)+and+resistant+(B10.D2)+mice.">Biological studies of E.L.4 lymphoma and C Leukemia in susceptible (C57BL) and resistant (B10.D2) mice.</a> Cancer Res. 24 (1498), 1785-1797 (1964).</li><li>Law, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+an+influence+affecting+growth+of+transplantable+leukemias+in+mice.">Characterization of an influence affecting growth of transplantable leukemias in mice.</a> Cancer Res. 4, 257-260 (1944).</li><li>Graham, J. D., McMahon Welch, C., Patchen, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+an+implanted+murine+myelogenous+leukemia+C1498.">Studies of an implanted murine myelogenous leukemia C1498.</a> Ohio J. Sci. 75 (4), 202-208 (1975).</li><li>Boyer, M. W., Orchard, P. J., Gorden, K. B., Anderson, P. M., Mclvor, R. S., Blazar, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dependency+on+intercellular+adhesion+molecule+recognition+and+local+interleukin-2+provision+in+generation+of+an+in+vivo+CD8++T-cell+immune+response+to+murine+myeloid+leukemia.">Dependency on intercellular adhesion molecule recognition and local interleukin-2 provision in generation of an in vivo CD8+ T-cell immune response to murine myeloid leukemia.</a> Blood. 85 (9), 2498-2506 (1995).</li><li>Labelle, J. L., Truitt, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+murine+NKT+cell+tumor+previously+described+as+an+acute+myelogenous+leukemia.">Characterization of a murine NKT cell tumor previously described as an acute myelogenous leukemia.</a> Leuk. Lymphoma. 43 (8), 1637-1644 (2002).</li><li>Bradner, W. T., Pindell, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+leukemia+as+a+screen+for+cancer+chemotherapeutic+agents.">Myeloid leukemia as a screen for cancer chemotherapeutic agents.</a> Cancer Res. 26 (4), 375-390 (1966).</li><li>Lin, J. M., Li, B., Rimmer, E., VanRoey, M., Jooss, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+the+anti-tumor+efficacy+of+a+GM-CSF-secreting+tumor+cell+immunotherapy+in+preclinical+models+by+cytosine+arabinoside.">Enhancement of the anti-tumor efficacy of a GM-CSF-secreting tumor cell immunotherapy in preclinical models by cytosine arabinoside.</a> Exp. Hematol. 36 (3), 319-329 (2008).</li><li>Robinson, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Flow Cytometry Methods. , (1993).</li><li>Xu, Y., McKenna, R. W., Wilson, K. S., Karandikar, N. J., Schultz, R. A., Kroft, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunophenotypic+identification+of+acute+myeloid+leukemia+with+monocytic+differentiation.">Immunophenotypic identification of acute myeloid leukemia with monocytic differentiation.</a> Leukemia. 20 (7), 1321-1324 (2006).</li><li>Hassan, K., Qureshi, M., Shafi, S., Ikram, N., Akhtar, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+myeloid+leukemia-FAB+classification+and+its+correlation+with+clinico-haematological+features.">Acute myeloid leukemia-FAB classification and its correlation with clinico-haematological features.</a> J. Pak. Med. Assoc. 43 (10), 200-203 (1993).</li><li>Quesenberry, P. J., Rappeport, J. M., Fountebouni, A., Sullivan, R., Zuckerman, K., Ryan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+normal+murine+hematopoiesis+by+leukemic+cells.">Inhibition of normal murine hematopoiesis by leukemic cells.</a> N.Engl.J. Med. 299 (2), 71-75 (1978).</li><li>Youn, J. I., Nagaraj, S., Collazo, M., Gabrilovich, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subsets+of+myeloid-derived+suppressor+cells+in+tumor-bearing+mice.">Subsets of myeloid-derived suppressor cells in tumor-bearing mice.</a> J. Immunol. 181 (8), 5791-5802 (2008).</li><li>Loder, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cell+development+in+the+spleen+takes+place+in+discrete+steps+and+is+determined+by+the+quality+of+B+cell+receptor-derived+signals.">B cell development in the spleen takes place in discrete steps and is determined by the quality of B cell receptor-derived signals.</a> J. Exp. Med. 190 (1), 75-89 (1999).</li><li>Robbins, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extramedullary+hematopoiesis+generates+Ly-6C(high)+monocytes+that+infiltrate+atherosclerotic+lesions.">Extramedullary hematopoiesis generates Ly-6C(high) monocytes that infiltrate atherosclerotic lesions.</a> Circulation. 125 (2), 364-374 (2012).</li></ol>

The authors declare that they have no competing financial interests.

In previous studies, the C1498 cell line was described as an inducer of acute granulocytic5, myelomonocytic6 or NKT7 cell leukemia. However, demonstrative data in the literature were either absent or incomplete. The protocol presented here uses different techniques, such as flow cytometry, immunofluorescence, MGG staining and cytochemical assays, to characterize cultured C1498 cells and to determine the nature of the leukemia that is induced in mice after they are injected.
When we phenotyped in vitro cultured C1498 cells after immunostaining and flow cytometry analyses were performed, we observed some limitations because these cells expressed few cell surface hematopoietic markers that have been previously described in the literature6,7. In agreement with our results, Labelle et al. did not observe the cell surface expression of mature TCR on C1498 cells using flow cytometry staining. However, they considered them to be a NKT cell line after they detected CD3&#949; and TCRV&#946;8.2 mRNAs7. We also observed the intracellular expression of TCRV&#946; chains and CD3&#949; molecules in most of the cells (&#62; 70%), but their hematopoietic lineages could not be determined because there was also concomitant intracellular expression of the Mac-3 molecule.
Myeloperoxidase, MGG staining and assessments to analyze functional esterases using cytochemistry demonstrated that the C1498 cell line had a myeloid origin and was composed of monoblasts and myeloblasts. These results were concordant with the percentage of Mac-3+ cells that were obtained in flow cytometry staining analysis. Although not quantitative, these steps represent key experiments to be performed. Indeed, they remain, so far, the best existing methods for characterizing the lineage and differentiation stage of hematopoietic cells that express no or few specific phenotypic markers.
Flow cytometry staining was helpful for demonstrating the development of acute leukemia in congenic mice after C1498 cells were intravenously injected. The CD45.2+ C1498 cells that infiltrated into the peripheral blood and various organs were isolated, and their frequencies were determined. Quantification was also performed to analyze inherent medullary and splenic cells after immunophenotyping. Limitations were encountered when the C1498 cell phenotype was examined in organs as they expressed few hematopoietic markers (only a few of them were B220+). To define the nature of the observed acute leukemia, May-Gr&#252;nwald Giemsa staining and an analysis of the activities of monocytic and granulocytic esterases were performed using bone marrow. The results showed that C1498 cells preserved their myeloblastic and monoblastic morphology and function, revealing the onset of myelomonocytic leukemia.
In consideration for the critical steps described in this protocol, particular attention should be given to pH when performing cytochemical reactions and MGG staining because errors in pH can lead to incorrect interpretations of results. For instance, &#945;-naphthyl butyrate esterase activity is specific to monocytic cells only at a pH of 6.0 because granulocytes and lymphocytes can also stain positive for this test at higher pH values. Fixating the cells is not recommended before performing MGG staining, and we showed that only CAF fixation provided satisfactory results when performing esterases cytochemical reactions using C1498 cells. To preserve the expression of the CD115 molecule and its detection by flow cytometry, all of the samples (e.g., blood, bone marrow, and spleen cells) should be kept on ice during the procedure. If no staining is observed in flow cytometry or/and immunofluorescence experiments, the reference of the antibodies, their storage recommendations and their dilutions should be checked. The references specified in the materials/equipment table have been selected for flow cytometry or immunofluorescence applications. The primary/secondary antibodies or their conjugated fluorophores might have lost their activity due to inappropriate storage (e.g., exposure to light or heat), improper dilution, extensive freezing/thawing or the use of contaminated buffers. Run positive controls to ensure that they are working properly. Use mouse bone marrow or spleen-derived cells that are known to express the proteins of interest. To avoid high background and non-specific staining, make sure that the cells are washed properly and kept at high humidity (for immunofluorescence) and that the antibodies are diluted as instructed. Use the same concentration and dilution for the isotype control antibody and the primary antibody to accurately determine the background level in the sample. For esterase cytochemistry experiments, the reagents can be tested by using positive and negative control slides containing purified mouse splenic granulocytic (Ly6G+) and monocytic (CD115+) cells.
The procedure described in this study showed that many of the leukemic features observed in mice after the injection of C1498 cells shared common hallmarks with human acute myelomonocytic leukemia11,12. The invaded leukemic cells resulted in a reduction of mature and immature (progenitors and precursors) medullary hematopoietic cells. C1498 cells are present at high frequency (&#62; 20%) in the peripheral blood, as are monocytic cells. Hepatomegaly and splenomegaly were observed to result from the infiltration of leukemic cells, and significant increases in B lymphocytes and myeloid cells were also observed to accompany splenomegaly. Thrombopenia was also observed when blood platelets numbers were estimated using a hematology analyzer.
It was shown, using in vitro experiments, that C1498 cells inhibit normal murine hematopoiesis by secreting soluble factors13. In several tumor mouse models, immature myeloid cells (including monocytic and granulocytic cells) have also been shown to migrate from the bone marrow to the spleen, where they inhibit anti-tumor specific T cell activation and proliferation14. Thus, the reduction in hematopoietic cells that was observed in the bone marrow could have resulted from either a deficiency in hematopoiesis and/or from their emigration. This latter mechanism could explain the presence of monocytosis in the peripheral blood or the observation of enlarged myeloid fractions in the spleen. It is also conceivable that these cells could have been derived from improved splenic hematopoiesis. Indeed, under steady-state conditions, some subsets of splenic B cells were identified as precursors of mature B lymphocytes15. Moreover, under inflammatory conditions, medullary stem and progenitors cells have been shown to relocate to the spleen to induce the production of mature monocytes16. This protocol does not allow us to draw conclusions regarding the mechanisms that are involved in the development of leukemia, and additional functional as well as molecular assays should be employed to do so. However, these data include detailed information about the clinical features of acute myelomonocytic leukemia and will help researchers to evaluate and understand the effects of new therapeutic agents.

Acute myeloid leukemia (AML) is characterized by the uncontrolled proliferation of hematopoietic myeloid cells that are blocked at different stages of maturation. This dysregulation can affect the granulocytic, monocytic, erythrocytic or megaryocytic differentiation pathways1. AML cells accumulate in the bone marrow, leading to impaired hematopoiesis, which results in thrombopenia, lymphopenia and anemia. The leukemic cells also invade the blood and non-lymphoid organs.
The C1498 mouse model has been used for decades as a model for acute leukemia since cancer cells were isolated from a leukemic 10-month-old C57BL/6 (H-2b) female mouse in 1941. The literature describes the invasion into the blood, hematopoietic organs (e.g., the spleen and lymph nodes) and non-hematopoietic organs (e.g., the liver, lungs, ovaries, and kidneys) by highly proliferative C1498 cells after they were injected via an intravenous, subcutaneous or intra-peritoneal route into susceptible mice2-4. However, this mouse model was reported to induce either granulocytic2,5 or myelomonocytic6 leukemia. More recently, a study published in 2002 described this type of cancer as murine NKT cell leukemia7. Thus, the literature differs concerning the nature of this C1498 cell line and the associated leukemia it induces in mice. These discrepancies are mainly due to a lack of detailed and updated published information about the cells and the leukemic disease in general because many studies were performed in the 1950 - 70&#39;s.
Here we provide a detailed protocol to describe how to characterize C1498 cells and analyze the nature of the leukemic disease that is induced by their intravenous injection into mice. The first section of this protocol is dedicated to a description of C1498 cells that have been cultured in vitro. Fluorescent antibodies directed against surface and intracellular hematopoietic markers were used to determine their phenotype using flow cytometry. The presence of myeloperoxidase was assessed using immunofluorescence microscopy, their hematopoietic lineage and differentiation stage were evaluated using cytochemistry to assess the activity of esterases, and May-Gr&#252;nwald Giemsa staining was performed. The C1498 cells were then injected into mice, and the acute leukemia disease that was induced is described in the second section of this manuscript. The same techniques were used to determine the frequencies and the phenotypes of leukemic and inherent cells in the bone marrow, peripheral blood, spleen and non-hematopoietic organs (the liver and lungs).
This protocol is highly reproducible, and the data presented here will help researchers to assess the effects of new therapeutic strategies. This leukemia mouse model has already been used to test immunotherapy approaches and different cancer chemotherapeutic drugs8,9. Their efficacy was evaluated by determining the evolution of tumor burden and survival rates. This protocol can be used to provide additional information about the distribution and subsistence of leukemic and other hematopoietic cell populations during treatment.

a detailed protocol for characterizing the murine c1498 cell line and its associated leukemia mouse model

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of acute leukemia. However, the nature of this disease has not been well documented in the literature. Here, we provide a technical protocol for characterizing C1498 cells in vitro and for determining the nature of the induced leukemia in vivo. The first part of this procedure is focused on determining the hematopoietic lineage and the stage of differentiation of cultured C1498 cells. To achieve this, multi-parametric flow cytometric staining is used to detect hematopoietic cell markers. Immunofluorescence microscopy, cytochemistry and a May-Gr&#252;nwald Giemsa staining are then performed to assess the expression of myeloperoxidase, the activity of esterases and cellular morphology, respectively. The second part of this protocol is dedicated to describing the leukemia disease that is induced in vivo. The latter can be achieved by determining the frequencies of leukemic and inherent cells in the blood, hematopoietic organs (e.g., bone marrow and spleen) and non-lymphoid tissues (e.g., the liver and lungs) using specific staining and flow cytometry analyses. The nature of the leukemia is then confirmed using May-Gr&#252;nwald Giemsa staining and staining for specific esterases in the bone marrow. Here, we present the results that were obtained using this protocol in age-matched C1498- and PBS-injected mice.

To characterize the C1498 mouse model, we proceeded with two major steps. First, the C1498 cells were characterized to determine their hematopoietic lineage and maturation stage in vitro (Figure 1). These cells were then injected into congenic mice, and the nature of the induced leukemic disease was assessed to determine different features: leukemic cell infiltration, their phenotype, a quantification of the hematopoietic cells (mature and progenitors/precursors) in bone marrow, the frequencies of C1498 cells and mature hematopoietic cells in the blood and an evaluation of organ swelling (in the spleen, liver, and lungs) and cellular composition.
To characterize the C1498 cell phenotypes in vitro, the cells were labeled with antibodies directed against molecules that are expressed by hematopoietic precursors and mature cells (Table 1), and the results were analyzed using flow cytometry. The C1498 cells were positive for cell surface expression of Mac-1 (CD11b/CD18) (&#8764; 7%), B220 (&#62; 25%), and they displayed intracellular expression of CD3&#949;, T-Cell Receptor (TCR) V&#946; chains and Mac-3 (Figures 2A and B). The cells were negative for the cell surface markers Ly6G, Ly6C, CD115, CD21/CD35, CD19, CD3, CD4, CD8, NK1.1, and pan-NK molecules and for the intracellular expression of CD4 and CD8 (data not shown). They were then examined for markers of hematopoietic stem cells and progenitors (Table 1). They were also negative for cell surface expression of CD117, CD34, Sca-1, CD150 and CD16/32 (data not shown). These leukemic cells were then tested to determine the expression of adhesion, antigen presentation and co-stimulatory molecules. The cells expressed the surface markers LFA-1 (CD11a/CD18), CD44, CD31 (PECAM-1), and H-2Db and were negative for MHC class II, CD80, CD86 and CD274 (data not shown). C1498 cells therefore expressed both myeloid (Mac-1, Mac-3) and lymphoid markers (B220, CD3, TCR).
To better characterize their hematopoietic lineage, myeloperoxidase expression was assessed using immunofluorescence microscopy. All of the cells were positive for the myeloperoxidase, which verified their myeloid origin (Figure 3A). A majority of the cells also stained positive for &#945;-naphthyl butyrate esterases (Figure 3B, left panel), and some of them stained for the naphthol AS-D chloroacetate esterases (black arrows) (Figure 3B, right panel). The results indicate that the cells contained mixtures of monocytic and granulocytic cells. After May-Gr&#252;nwald Giemsa staining was performed, the C1498 cells were observed to display a blast-like morphology with a high nucleo-cytoplasmic ratio, 3 to 5 nucleoli in the nucleus, a perinuclear halo, numerous vacuoles and a basophilic cytoplasm (Figure 3C). Thus, the C1498 cell line is composed of monoblasts and myeloblasts.
The C1498 cells (CD45.2+) were then intravenously injected into CD45.1+ mice. The mice succumbed 17 to 19 days after the cells were injected. These mice were sacrificed so that their leukemia type could be analyzed before they died from the disease. The control mice, which were injected with PBS, were analyzed at the same time points for comparison. The C1498 cell-injected mice displayed massive infiltration of C1498 cells into their bone marrow, as demonstrated by the blast-like appearance of the cells after May-Gr&#252;nwald Giemsa staining was performed (Figure 4A). They also preserved their monocytic and granulocytic phenotypes (Figure 4B and C), demonstrating an accumulation of monoblastic and myeloblastic cells that is characteristic of acute myelomonocytic leukemia.
To determine whether medullary hematopoietic cells numbers were lower following leukemic cells invasion, CD45.2+ C1498 cells, B lymphocytic, monocytic and granulocytic populations (including progenitors, precursors and mature cells), were quantified using immunofluorescent staining and multi-parametric flow cytometry analysis. Leukemic cells represented 16 to 36% of the hematopoietic cells (data not shown). The other cell types were all present in significantly lower numbers in the C1498-injected mice than in the PBS-injected mice (by 5-fold on average for B cell subsets, 4-fold on average for granulocytic cells and 3-fold on average for monocytic subsets) (Figure 5A to C).
An investigation of the frequencies of mononuclear cells in leukemic and control mouse blood samples showed that they contained a comparable percentage of lymphocytes (Figure 6A) but a higher frequency of monocytic and leukemic cells. These characteristics are representative of acute myelomonocytic leukemia11 (Figure 6B).
Among the other features of acute myelomonocytic leukemia12, the C1498-injected mice presented with swollen livers (hepatomegaly), lungs and spleens (splenomegaly) (Figure 7A). Various frequencies of CD45.2+ C1498 cells were detected in these organs using immunofluorescent staining and flow cytometry analysis (Figure 7B). As splenomegaly can result from high numbers of infiltrated monocytes, we also estimated the proportions of splenic populations. The numbers of cells in the B lymphocytic, monocytic and granulocytic cell fractions were significantly larger, by an average of 2-fold, 2.5-fold and 3-fold, respectively, in leukemic spleens than in control spleens (Figure 7C).
<img alt="Figure 1" src="/files/ftp_upload/54270/54270fig1.jpg" /> 
Figure 1. Schematic Representation of the Protocol Set Up for Characterizing in vitro Cultured C1498 Cell Lines and in vivo Descriptions of Acute Leukemia. The hematopoietic lineage and the differentiation stage of tissue-cultured C1498 cells were first determined. C1498 cells were then injected into congenic mice to induce the development of acute leukemia. The isolation of bone marrow, peripheral blood, spleen, liver and lung tissues was performed to determine the frequencies, phenotypes and morphological changes after the C1498 cells infiltration. IV: Intravenous MGG: May-Gr&#252;nwald Giemsa. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54270/54270fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54270/54270fig2.jpg" /> 
Figure 2. Phenotypic Analysis of C1498 Cells after in vitro Culture. Representative flow cytometry dot plots and histograms of cell surface (A) and intracellular (B) C1498-expressed molecules that were associated with hematopoietic mature cell differentiation are shown. C1498 cells were harvested from cultures, washed and labeled using fluorescent antibodies that were specific for the cell surface CD11b, CD18 and B220 markers or their isotype controls. For intracellular staining, the cells were fixed, permeabilized and labeled using antibodies directed against Mac-3, CD3&#949;, and a common epitope of the TCR (T-Cell Receptor) V&#946; chain or their isotype controls. Analyses were performed using gating with live cells. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54270/54270fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54270/54270fig3.jpg" /> 
Figure 3. Functional and Morphological Characterization of Cultured C1498 Cells. C1498 cells were harvested from cultures and centrifuged on slides for microscopy. (A) Staining for myeloperoxidase expression was performed using immunofluorescence. (B) Cytochemical reactions were used to analyze the &#945;-naphthyl butyrate esterase (NBE) and naphthol AS-D chloroacetate esterase (CAE) activities in C1498 cells. Cells were considered to be positive for each label when brown and red-purple, large cytoplasmic granules, respectively, were observed. (C) May-Gr&#252;nwald Giemsa (MGG) staining of C1498 cells. For each staining experiment, the microscopy objective magnification is indicated. Each image is representative of three separate experiments. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54270/54270fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54270/54270fig4.jpg" /> 
Figure 4. Bone Marrow Morphologies in PBS- and C1498-injected Mice. Bone marrow cells were isolated from PBS- and C1498 cell-injected mice and centrifuged onto slides for microscopy. (A) May-Gr&#252;nwald Giemsa (MGG) staining. (B) &#945;-naphthyl butyrate esterase (NBE) and (C) naphthol AS-D chloroacetate esterase (CAE) functions were evaluated using cytochemistry. In panel A, the band (immature) or segmented (mature) neutrophils are less visible in the bone marrow of the C1498-injected mice than the PBS-injected mice. Panel B and C indicate that there was an accumulation of monocytic and granulocytic cells in the leukemic bone marrow compared to the numbers observed in the control bone marrow. All microscopic analyses were performed using a 100X magnification objective. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54270/54270fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54270/54270fig5.jpg" /> 
Figure 5. Quantitative Analysis of Medullary Populations in PBS- and C1498-injected Mice. Bone marrow cells were isolated from PBS- and C1498 cell-injected mice and estimated after cell counting was performed. The frequencies of the different cell populations were determined after immunostaining and live cell gated flow cytometry analysis. (A) The B cell subsets included CD19+B220+cells in stages from pro-B cells to mature B lymphocytes (B) granulocytic cells in the CD3- and CD11b+Ly6G+ lineages, which included precursors and immature and mature granulocytes. (C) The monocytic subsets were defined as CD3-CD115+ and included cells in the progenitor to mature monocyte stages. n = 7 mice/group, and the data are presented as histograms showing the means &#177; SEM. ***, p&#60; 0.0001 and **, p&#60; 0.01, unpaired Student&#39;s t-test comparing PBS- and C1498-injected mice. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54270/54270fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54270/54270fig6.jpg" /> 
Figure 6. Blood Analysis of Mononuclear Cell Subsets in PBS- and C1498-injected mice. Representative flow cytometry dot plots of (A) T and B lymphocyte percentages, which were respectively defined as CD3+ and B220+ cells in PBS- and C1498 cell-injected mice. (B) Monocytic cell frequencies in C1498 leukemic and control (PBS) mice were determined by analyzing CD115+Ly6C- and CD115+Ly6Chigh cells. The analysis was performed by gating live cells. To compare leukemic and control mice, CD45.2+ C1498 cells were excluded. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54270/54270fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/54270/54270fig7.jpg" /> 
Figure 7. Estimation of Splenic Populations in Leukemic and Control Mice. (A) Representative photographs of liver, lung and spleen swelling in leukemic mice compared to control mice. Spleens were collected and weighed, and splenocytes were counted following tissue disruption. (B) Histogram representing leukemic cell frequencies in different organs after immunostaining was performed for CD45.2+ cells and the results were analyzed using flow cytometry. (C) Estimations of splenic B, granulocytic and monocytic cell numbers after immunostaining and flow cytometry analysis gating were performed to identify live CD19+B220+, CD3-CD11b+Ly6G+, CD3-CD11b+Ly6C- and CD3-CD11b+Ly6Chigh cells. The scale bars shown for the lungs, spleens and livers indicate 1 cm. n = 5 - 8 mice/group, and the data are represented in histograms as the means&#160;&#177; SEM. *, p&#60; 0.05; **, p= 0.0033, unpaired Student&#39;s t-test comparing PBS- and C1498-injected mice. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54270/54270fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Cell Type</td>
			<td>Membrane or Intracellular Molecules&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Precursors and Mature cells</td>
			<td></td>
		</tr>
		<tr>
			<td>NK cells</td>
			<td>NK1.1+, pan-NK+</td>
		</tr>
		<tr>
			<td>NKT cells</td>
			<td>NK1.1+, pan-NK+, TCR Vbeta+(8.2), CD3+</td>
		</tr>
		<tr>
			<td>T lymphocytes</td>
			<td>TCR Vbeta+, CD3+, CD4+, CD8+</td>
		</tr>
		<tr>
			<td>B cells precursors and B lymphocytes</td>
			<td>B220+, CD19+, CD21/35+&#160;</td>
		</tr>
		<tr>
			<td>granulocytic precursors and granulocytes</td>
			<td>Ly6G+, Mac-1+, CD11b+</td>
		</tr>
		<tr>
			<td>monocytic precursors and monocytes/macrophages</td>
			<td>CD11b+, Mac-1+, Mac-3+, CD21/35+, CD115+, Ly6Chi</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Progenitors</td>
			<td></td>
		</tr>
		<tr>
			<td>multipotent progenitors</td>
			<td>CD117+ Sca-1+ CD34+ (Lin- CD150-)</td>
		</tr>
		<tr>
			<td>lymphoid-primed multipotent progenitors&#160;</td>
			<td>CD117hi Sca-1hi CD127+ (Lin- )</td>
		</tr>
		<tr>
			<td>common lymphoid progenitors&#160;</td>
			<td>CD117lo Sca-1lo CD127+ (Lin- )</td>
		</tr>
		<tr>
			<td>common myeloid progenitors&#160;</td>
			<td>CD16/32lo CD117+ CD34int (Lin- Sca-1-)</td>
		</tr>
		<tr>
			<td>granulocyte-macrophage progenitors&#160;</td>
			<td>CD16/32hi CD117+ CD34hi (Lin- Sca-1-)</td>
		</tr>
		<tr>
			<td>megakaryocyte-erythroid progenitors&#160;</td>
			<td>CD16/32lo CD117+ CD34lo (Lin- Sca-1-)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hematopoietic stem cells&#160;</td>
			<td>CD117+ Sca-1+ CD150+ (Lin- CD34-)</td>
		</tr>
	</tbody>
</table>
Table 1. Markers of Hematopoietic Cell Lineages and Differentiation. 
CD: cluster of differentiation; Lin: markers of mature cells; lo: low expression; hi: high expression; int: intermediate expression; NK: natural killer cells; TCR: T-cell receptor.

Watch this Scientific Journal Video about A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model at JoVE.com

A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

This manuscript provides a technical procedure that can be used to characterize C1498 cell cultures in vitro and the acute leukemia induced in mice after their injection. Phenotypic and functional analyses are performed using flow cytometry, immunofluorescence microscopy, cytochemistry and May-Gr&#252;nwald Giemsa staining.

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of ...

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19.8K Views.  INSERM, UMR-S-1172, Centre de Recherche Jean-Pierre Aubert (JPARC). This manuscript provides a technical procedure that can be used to characterize C1498 cell cultures in vitro and the acute leukemia induced in mice after their injection. Phenotypic and functional analyses are performed using flow cytometry, immunofluorescence microscopy, cytochemistry and May-Grünwald Giemsa staining.

Video: A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

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Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study end-points. Thus, studies focused on elucidating mechanisms of late-stage de novo metastasis are compromised, as are studies examining efficacy of anti-cancer therapies targeting mediators of metastasis in the adjuvant setting. Numerous murine mammary cancer models have been developed via targeted expression of dominant oncoproteins to mammary epithelial cells yielding models variably mimicking histopathologic and transcriptome-defined breast cancer subtypes common in women1. While much has been learned regarding the biology of mammary carcinogenesis with these models, their utility in identifying molecules regulating growth of late-stage metastasis are compromised as mice are typically euthanized at earlier time points due to significant primary tumor burden. Moreover, since a significant percentage of women diagnosed with breast cancer receive adjuvant therapy after surgical resection of primary tumors and prior to presence of detectable metastatic disease, preclinical models of de novo metastasis are urgently needed as platforms to evaluate new therapies aimed at targeting metastatic foci. To address these deficiencies, we developed a murine model of de novo mammary cancer metastasis, wherein primary mammary tumors are surgically resected, and metastatic foci subsequently develop over a 115 day post-surgical period. This long latency provides a tractable model to identify functionally significant regulators of metastatic progression in mice lacking primary tumor, as well as a model to evaluate preclinical therapeutic efficacy of agents aimed at blocking functionally significant molecules aiding metastatic tumor survival and growth.

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

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A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

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Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival. Not only is the total leukemic population, reflected by cells harboring a leukemia associated immune-phenotype (LAIP), associated with clinical outcome, but so is the immature low frequency subpopulation of leukemia stem cells (LSC), both of which can be monitored through flow cytometry MRD or MRD-like approaches. The availability of sensitive assays that enable detection of residual leukemia (stem) cells on the basis of disease-specific or disease-associated features (abnormal molecular markers or aberrant immunophenotypes) have drastically improved MRD assessment in AML. However, given the inherent heterogeneity and complexity of AML as a disease, methods for sampling bone marrow and performing MRD and LSC analysis should be harmonized when possible. In this manuscript we describe a detailed methodology for adequate bone marrow aspirate sampling, transport, sample processing for optimal multi-color flow cytometry assessment, and gating strategies to assess MRD and LSC to aid in therapeutic decision making for AML patients.

Detection of minimal or measurable residual disease (MRD) is an important prognostic biomarker for refining risk assessment and predicting relapse in acute myeloid leukemia (AML). These comprehensive guidelines and recommendations with best practices for consistent and accurate identification and detection of MRD, may aid in making effective AML treatment decisions.

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Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

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Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. The power of this preparation lies in its ability to mimic many aspects of the in vivo conditions of tumors while being fast, cheap, and versatile enough to allow relatively high-throughput screening. The spheroid culture conditions can recapitulate the physico-chemical gradients in a tumor, including the increasing extracellular acidity, increased lactate, and decreasing glucose and oxygen availability, from the spheroid periphery to its core. Also, the mechanical properties and cell-cell interactions of in vivo tumors are in part mimicked by this model. The specific properties and consequently the optimal growth conditions, of 3D spheroids, differ widely between different types of cancer cells. Furthermore, the assessment of cell viability and death in 3D spheroids requires methods that differ in part from those employed for 2D cultures. Here we describe several protocols for preparing 3D spheroids of cancer cells, and for using such cultures to assess cell viability and death in the context of evaluating the efficacy of anticancer drugs.

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The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by introducing specific mutations in oncogenes and tumor suppressor genes, using clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome editing technology in mice. This technology allows us to accurately mimic the genetic changes that occur in human cancers using mice. By genetically transforming murine primary cells, we can better study cancer development, progression, treatment, and diagnosis. In this study, we used Cre-inducible Cas9 mouse tongue epithelial cells to enable genome editing using adeno-associated virus (AAV) in vitro. Specifically, by altering KRAS, p53, and APC in normal tongue epithelial cells, we generated a murine head and neck cancer (HNC) cell line in vitro,which is tumorigenic in syngeneic mice. The method presented here describes in detail how to generate HNC cell lines with specific genomic alterations and explains their suitability for predicting tumor progression in syngeneic mice. We envision that this promising method will be informative and useful to study tumor biology and therapy of HNC.

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The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

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Chimeric antigen receptor (CAR)-modified immune cell therapy has become an emerging treatment for cancers and infectious diseases. NK-based immunotherapy, particularly CAR-NK cell, is one of the most promising 'off-the-shelf' development without severe life-threatening toxicity. However, the bottleneck for developing a successful CAR-NK therapy is achieving sufficient numbers of non-exhaustive, long-lived, 'off-the-shelf' CAR-NK cells from a third party. Here, we developed a new CAR-NK expansion method using an Epstein-Barr virus- (EBV) transformed B cell line expressing a genetically modified membrane form of interleukin-21 (IL-21). In this protocol, step-by-step procedures are provided to expand NK and CAR-NK cells from cord blood and peripheral blood, as well as solid organ tissues. This work will significantly enhance the clinical development of CAR-NK immunotherapy.

Natural Killer NK and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line

Here, we present a method to expand peripheral blood natural killer (PBNK), NK cells from liver tissues, and chimeric antigen receptor (CAR)-NK cells derived from peripheral blood mononuclear cells (PBMCs) or cord blood (CB). This protocol demonstrates the expansion of NK and CAR-NK cells using 221-mIL-21 feeder cells in addition to the optimized purity of expanded NK cells.

Chimeric antigen receptor (CAR)-modified immune cell therapy has become an emerging treatment for cancers and infectious diseases. NK-based immunotherapy, ...