**2. Material and methods**

#### **2.1 Mast cell (effector) development in methylcellulose and maintenance in suspension culture**

A colony forming unit (CFU) -Mast was produced in vitro by our modified method (Özdemir, 2007, 2011). From several discarded patient samples, human bone marrow (BM) mononuclear cells (≥5x104) were obtained and suspended in 0.3 ml Iscove's Modified Dulbecco's Medium (IMDM) containing %1 Fetal Bovine Serum (FBS) after Ficoll (Sigma, St. Louis, MO). Cells were put into 3 ml serum-free methylcellulose medium [MethoCult™ SFBIT H4236, StemCell Technologies, British Columbia, Canada], supplemented with 200 ng/ml of SCF, 50 ng/ml of IL-6 and 1ng/ml of IL-3 (only at the beginning). All cytokines were purchased from Biosource, Camarillo, CA. We inoculated 0.3 ml of the mixed medium in the 12-well plate with a 16 gauge blunt needle and placed into an incubator. Every two weeks, the cells were fed with 0.3 ml new medium including 100 ng/ml of SCF and 50 ng/ml of IL-6. Thirty or more cells were scored as CFU-Mast in situ on an inverted microscope after 4 weeks. Before culturing in suspension, MCs are retrieved from only CFU-Mast`s in the medium and dissolved with >2-fold volume of phosphate buffer solution including FBS %10 at 6th weeks. Cells are centrifuged at 250xG for 5 minutes. They are suspended and cultured in complete IMDM supplemented with 100 ng/ml of SCF, 50 ng/ml of IL-6 and 2% FBS in 25cm2 flask up to 8th weeks. The culture was started with ≤104 cells/ml and accumulated up to ≤2x106 cells/ml. The suspension culture was hemi-depleted every week and supplemented with 100 ng/ml of SCF and 50 ng/ml of IL-6.

#### **2.2 Staining for verification of effector mast cells**

4 Advances in Cancer Therapy

pathway mediators such as FasL and granzyme B expressions were detected in mice and cultured MCs; respectively (Wagelie-Steffen, 1998; Leskinen, 2003; Kataoka, 2004; Pardo, 2007; Heikkilä, 2008). Thus, except for perforin (Pardo, 2007), MCs indeed have been proven to have all components of short and long-term cell-mediated cytotoxicity, which consist of the secretory pathway (via soluble TNF-α, chymase, serine proteases granzyme-B/-H), and non-secretory pathway (the death receptors Fas L and membranous TNF-α) (Özdemir, 2006,

Nonetheless, some researchers still consider MC as an enhancer of tumor development through their angiogenic effects, causing invasiveness and metastasis of tumor tissue (Özdemir, 2006). Some MC mediators such as heparin, IL-8 and tryptase are known to be responsible for angiogenesis (Ribatti, 2000). Yet, neither these mediators are the only known elements responsible from neoangiogenesis, nor are MCs the only resource. MCs also have a vast array of mediators, some of which have promoting, and others inhibitory effects on angiogenesis besides malignancies (Özdemir, 2006). The same researchers consistently based their theories on pathological specimen observations, showing an association between increased MCD and the worst prognosis in some cancers such as endometrial cancer, leukemia as well as lymphomas (Ribatti, 2009; Molin, 2002). Our correspondences against this conviction have been well documented in recent literature

As summarized above, in this chaotic literature environment, our aim in this study was to investigate human MCC against NK- and lymphokine activated killer (LAK)-sensitive/ resistant human leukemia-lymphoma cells in short and long term coincubations by our

A colony forming unit (CFU) -Mast was produced in vitro by our modified method (Özdemir, 2007, 2011). From several discarded patient samples, human bone marrow (BM) mononuclear cells (≥5x104) were obtained and suspended in 0.3 ml Iscove's Modified Dulbecco's Medium (IMDM) containing %1 Fetal Bovine Serum (FBS) after Ficoll (Sigma, St. Louis, MO). Cells were put into 3 ml serum-free methylcellulose medium [MethoCult™ SFBIT H4236, StemCell Technologies, British Columbia, Canada], supplemented with 200 ng/ml of SCF, 50 ng/ml of IL-6 and 1ng/ml of IL-3 (only at the beginning). All cytokines were purchased from Biosource, Camarillo, CA. We inoculated 0.3 ml of the mixed medium in the 12-well plate with a 16 gauge blunt needle and placed into an incubator. Every two weeks, the cells were fed with 0.3 ml new medium including 100 ng/ml of SCF and 50 ng/ml of IL-6. Thirty or more cells were scored as CFU-Mast in situ on an inverted microscope after 4 weeks. Before culturing in suspension, MCs are retrieved from only CFU-Mast`s in the medium and dissolved with >2-fold volume of phosphate buffer solution including FBS %10 at 6th weeks. Cells are centrifuged at 250xG for 5 minutes. They are suspended and cultured in complete IMDM supplemented with 100 ng/ml of SCF, 50 ng/ml of IL-6 and 2% FBS in 25cm2 flask up to 8th weeks. The culture was started with ≤104 cells/ml and accumulated up to ≤2x106 cells/ml. The suspension culture was hemi-depleted

established flow cytometric (FCM) cytotoxicity methods (Özdemir, 2003, 2007, 2011).

**2.1 Mast cell (effector) development in methylcellulose and maintenance in** 

every week and supplemented with 100 ng/ml of SCF and 50 ng/ml of IL-6.

2007, 2011).

(Özdemir, 2006).

**2. Material and methods** 

**suspension culture** 

Verification of MCs was done by May-Grunwald-Giemsa, Wright-Giemsa, acid Toluidine Blue staining and immunophenotyping on FCM. In brief, a colony was lifted with Eppendorf micropipette and spun down at 600 rpm for 5 minutes in the 4th and 6th week. Viability was checked with a trypan blue exclusion test. Cells from colonies were stained with May-Grunwald-Giemsa and Wright-Giemsa for verification purposes (Fig.1A1-4). MCs were fixated with a Carnoy solution and incubated for 2 minutes with acid toluidine blue to confirm their tryptase content as well. Furthermore, MCs were immunophenotyped for all related markers in FCM at 4th-8th weeks (Fig.1B1-3, Table 1). All monoclonal antibodies (mAb) were purchased from Immunotech, Inc. (Westbrook, ME).

Fig. 1. A1- 4. Wright- Giemsa slides are showing conjugate formation between mast cell and both tumor cells (effector-target doublets). A1-2 show conjugate formation between mast and Daudi cells. A3-4 depict conjugate formation between mast and Raji cells.

Fig. 1. B1- 3. Phenotyping of 4- week- old human bone marrow -derived mast cells on flow cytometry is shown in a representative sample. B1 shows CD117 (c-kit) expression vs. SS (granularity) of mast cells. B2 demonstrates ≥98% of cells already stained with CD117 and became CD34 negative. B3 illustrates 93% of cells stained with CD33 and 76% of cells were positive for CD49d. [The human effector (mast) cells produced from bone marrow were absolutely negative for CD19 in this study.]

May Mast Cells Have Any Effect in New Modalities of Cancer Treatment? 7

Fig. 1. C1- E3: Target cell (DAMI) death is shown by new flow cytometric assay at 24h and

alone/coincubation samples using target cell labeling (DIOC18) and cell size characteristics (FS) is shown in histograms at the first row. Spontaneous death and cytotoxicity evaluation of the cell populations alone or in co-incubation samples are shown with the rectilinear boxes of related histograms at the second and third rows. C1 shows location of effector-mast cell. Since effector cells are not pre-stained with DIOC18, they are DIOC18 negative and in a different area (Region G). C2 and C3 demonstrate the change in viability of effector cell alone population (DIOC18−/PI− viable cells: 94% and 91%; respectively) at 24h/48h. Spontaneous deaths in these effector alone samples are 6% and 9%; respectively. These histograms are obtained after gating on region G of a corresponding sample. D1 shows the target-DAMI cell population from target alone tube. As expected, target cells are very wellmarked with DIOC18 (Region J). D2 and D3 depict the viability of the target population alone (DIOC18+/PI− viable cells: 96% and 95%; respectively) at 24h and 48h. Spontaneous deaths in these samples are 4% and 5%; respectively. These histograms are obtained after gating on region J of a corresponding sample. E1 obtained from an experiment where mast cells are coincubated with DIOC18-positive DAMI cells at 2:1 effector/ target ratio. In this co-incubation sample; effector/target gatings are defined according to the target/effector alone (control) tubes. E2 depicts decrease in viability of previously gated target cells in J region of a corresponding co-incubation sample, 7% of them are stained with PI+ indicating necrotic killing (Region L2). In this representative sample, DAMI cell viability slightly decreased from 96% in the control to 93% after 24h co-incubation. The 4% of spontaneous death increased up to 7% with cytotoxicity mediated by mast cells during 24h co-incubation. E3 shows obvious decrease in viability of previously gated target DAMI cells, as 26% of them are dead. In this representative sample, DAMI cell viability further decreased from 95% to 74% throughout 48h co-incubation. The necrotic populations increased from a corresponding 5% of spontaneous

48h in a representative sample. Identification of effector and target cells in

death to 26% kill mediated by mast cell cytotoxicity in the co-incubated sample.


Table 1. The phenotypic characterizations of 4- to 8- week-old human bone marrow- derived mast cells by flow cytometry in representative samples are shown.
