Öner Özdemir

*Ministry of Health, İstanbul Medeniyet University Göztepe Research / Training Hospital, Kadköy Republic of Turkey* 

#### **1. Introduction**

Multifunctional mast cells (MC) have been recently reported as effectors in the human innate and even adaptive immune system, besides their known roles in allergic disorders. First in vivo observations in the 1950`s suggested their possible role as anti-tumor cells around certain solid tumors and questioned their interactions with tumor cells (Prior, 1953). Later, in vitro murine mast cell cytotoxicity (MCC) against murine tumor cells was described in 1981 (Henderson, 1981; Ghiara, 1985; Richards 1988). However, to the best of our knowledge, there is no reported data on in vitro human MCC against human tumor cells. Current in vivo observations and implications from human pathological specimens are also very controversial. Moreover, there is still difficulty in obtaining and maintaining human MCs in cultures since they have low expansion potential. These facts have hampered in vitro human MC studies up to the last decades of the 20th century. The recent use of methylcellulose media for in vitro human MC cultures increased the knowledge and data strongly supporting an increasing role of MC as effector elements of innate immunity (Leskinen, 2003; Marshall, 2004; Della Rovere, 2009; Özdemir, 2007, 2011).

Ambiguous and mounting evidence also indicates that MCs accumulate around tumors and could either promote or inhibit tumor growth, most likely depending on environmental conditions. Presently, believers in the inhibitory role of MCs assume them to be inhibitors of tumor development through their cytotoxic pro-necrolytic/-apoptotic granules (Wagelie-Steffen, 1998; Leskinen, 2003; Kataoka, 2004; Pardo, 2007; Heikkilä, 2008). In fact, the MC has been long believed to have natural cytotoxicity against murine TNF-α sensitive tumor cells in the long term incubations (>24h), by either TNF-α dependent or independent pathways. TNF-α independent pathways like cathepsin G, NO, serine proteases, peroxidases, H2O2 etc. were also assumed to contribute to MCC (Henderson, 1981; Ghiara, 1985; Richards 1988). Moreover, in vitro murine MCC has been well demonstrated against TNF-α-sensitive murine WEHI-164 and L929 tumor cells. Murine MCC seems to be different from natural killer (NK) cytotoxicity by means of acting in long term against unusual targets such as WEHI-164 and L929 with different mediators like peroxidases (Henderson, 1981; Ghiara, 1985; Richards 1988). Moreover, the last decade of research demonstrates that MC granules have pro-apoptotic characteristics (Wagelie-Steffen, 1998; Leskinen, 2003; Kataoka, 2004; Pardo, 2007; Heikkilä, 2008). Chymase was shown to induce apoptosis, and apoptotic

May Mast Cells Have Any Effect in New Modalities of Cancer Treatment? 5

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

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

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.]

and Daudi cells. A3-4 depict conjugate formation between mast and Raji cells.

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

(mAb) were purchased from Immunotech, Inc. (Westbrook, ME).

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, 2007, 2011).

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 (Özdemir, 2006).

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