**4. Discussion**

14 Advances in Cancer Therapy

**Total Killing** 

**Daudi** 7% 50% 57 % 23% 40% 63 % **Raji** 8 % 18% 26 % 27% 40% 67 %

Table 2D. The distribution of mean killing (%, PC) measured by FCM-MCMCA in human LAK-sensitive tumor cells at 2:1 ratio on different coincubation times in a representative

In the literature, MCC did not seem to be very effective against some types of LAK-sensitive tumor cells, such as K562 and YAC-1 (Henderson, 1981; Ghiara, 1985; Richards 1988).Consistently, there was not significant killing (18% killing at most) in NK-/LAKsensitive K562 cells even with 5:1 ratio at 48h (Table 2A,C). These findings supported wellknown resistance to MCC by some LAK-sensitive cells. However, human MCC for first time was found to be very effective against different type of LAK-sensitive cells, such as Daudi and Raji, in this study. As shown in these studies, most of the total killing of both human target cells was necrotic kill and it increased over time. Interestingly, Raji killing, especially necrotic, apparently maximized at 24h, even though Daudi cell killing stayed almost stable and peaked at 12h. Both LAK-sensitive Daudi and Raji cell death were statistically significant between 2h-48h, compared to spontaneous killing. In vitro human MCC against

Moreover, in this study the FCM-MCMCA method allowed us to separate cytotoxic killing into different stages, early and late apoptotic (necrotic) kill. And distribution of apoptotic type killing according to the cell lines at 12h and 24h was shown at 2:1 ratio (Table 2D). Early apoptotic cell death up to 46% was also detected in the representative samples (Fig.1H2,I2), indicating the role of pro-apoptotic components of MC granules in human

Although at 2h/12h/18h there was some killing, statistically significant killing in LAKresistant cells such as Meg-01 and HL-60 cells was demonstrated with both 2:1 and 5:1 ratios at 48h (Table 2A,C). In HL-60 cells, there was a statistically significant degree of kill at 18h (ratio 5:1) and 24h (ratio 2:1) as well. Moreover, the AML patient samples seemed to be very resistant to MCC. In patient cells, there was a fair amount of killing (≤25 %) but was not statistically significant (Table 2A,C). Insignificant killing in patient samples and LAKresistant cells, probably due to small number of sample, but it was noticeably important. This is the first study demonstrating human MCC against some human LAK-resistant cells

After 2h/12h coincubations, neither at 2:1 nor at 5:1 ratios was there significant killing in any experiments except for Daudi and Raji cells. In LAK-sensitive Daudi/Raji cells, statistically significant death at different rates was demonstrated and remained significant from 2h-48h coincubation (Table 2B-C). Concurrently, short term MCC (≤18h) was observed in patient samples as well as DAMI/HL-60/Meg-01 cells in this study, although MCC in the long term is well-known. We even observed some degree of cytotoxicity in very short-term incubation (2h) in DAMI/Meg-01 cells, although there was not statistically significant killing. The killing detected between 2h-48h in all LAK-resistant cells including patients'

**Percentage Based Human Mast Cell- Mediated Cytotoxicity (%)**  12h 24h

> **Early Apoptotic**

**Late Apoptotic** 

**Total Killing** 

**Cell Types** 

sample.

MCC.

too.

**Early Apoptotic** 

**Late Apoptotic** 

LAK-sensitive cells at different ratios/times was shown in table 2B-C.

This study first of all demonstrated human MCC against human tumor targets by two different approaches that were very comparable to CRA. These are established and reliable methods elucidating MCC by a two- and three-color FCM assays using DIOC18 and mAb target-cell marking, respectively. Our research experiences with these methods have been recently published (Özdemir, 2003, 2007, 2011).

As mentioned above, murine MCC against lysis-sensitive murine tumor cell lines (WEHI– 164/L929) in long term was previously well demonstrated (Henderson, 1981; Ghiara, 1985; Richards 1988). Nevertheless, to the best of our knowledge, this is the first study using human BM-derived effector MCs against human tumor cells to show and verify short- and long-term human MCC in vitro. Even significant kill in different LAK-sensitive/-resistant cells was also demonstrated for the first time. MCC was shown to be effective against different types of NK-/LAK-sensitive Daudi/Raji cells, but not to K562 cells or LAKresistant cells. These findings are consistent with earlier murine studies demonstrating murine MCC against lysis-sensitive cells. Like murine cells, Daudi/Raji are also known to be somewhat lysis (TNF-α)-sensitive, and this explains statistically significant and mostly

May Mast Cells Have Any Effect in New Modalities of Cancer Treatment? 17

Taskinen, 2010;). Amazingly, in some studies, human tumors in which MCD is supposed to correlate with a good prognosis include HL, diffuse large B-cell lymphoma, and follicular lymphoma (Koster, 2005; Hedström, 2007; Rygoł, 2007;Ribatti, 2009;). Although it is hard to explain these conflicting results above and in the literature, they may be associated with different methodologies used in studies such as timing of biopsy (e.g. doing in early instead of late stage of tumor), the tumor type, as well as environmental factors surrounding that tumor (Özdemir, 2006). Only observing increased MCD in various tumors with good or bad prognosis on pathological specimens seems to be far behind to explain the real role of MCs. The net effect of MCs on tumor growth, therefore, is likely to be the result of multiple interactions between MC, tumor, and associated inflammatory cells with their signaling pathways, and adjacent stromal cells such as vascular endothelium and fibroblasts (Ribatti,

Consequently, antiangiogenic strategies have recently become an important therapeutic modality for solid tumors. And MCs have been thought of as a new target for the adjuvant treatment of tumors through the selective inhibition of angiogenesis, tissue remodeling and tumor promoting molecules. Preliminary studies with cromolyn in mice mammary adenocarinoma and pancreatic cancer therapy was ineffective. Although some therapeutics like alemtuzumab were also given as an example of antiangiogenic treatment success, it is known as an anti-myeloid cell antiangiogenic agent used for the treatment of ovarian cancer, not an anti-MC agent (Ruan, 2009). Beside MCs and myeloid cells, Tie2-expressing monocytes and vascular leukocytes have recently been shown as new targets in the regulation of tumor associated angiogenesis (De Palma, 2007). In the age of targeted therapy, studies of the targeting MCs` role in cancer might have direct clinical consequences and should be further elucidated via the use of histopathological and complex biological

Since the aim of this study was basically to show MCC using human effector/target cells, the mechanisms and specificity of human MCC were not evaluated in this study. Our studies still continue studying MC-tumor cell relations and the characteristics of MCC on human pathological biopsy specimens. We hypothesize that human MCC may happen thru non-secretory pathway (cell-to-cell contact) of membranous TNF-α/Fas L, and secretory pathway via released mediators like soluble TNF-α, granzymes and chymase. Simply, MCC happens via pro-apoptotic/-necrolytic granules of MCs in the short term, as well as through its membranous components (non-secretory pathway of MCC) in the long term as shown in this study. Consistently, observing effector-target doublets on Wright-Giemsa slides of this study might suggest the importance of cell-to-cell contact in this process. The mechanisms with characteristics of human MCC against human tumor cells, as well as the role of MC

Our findings in this study verify possible anti-tumor role of MC as a contributory effector cell to NK and other cytotoxic cells in immunosurveillance within human innate immunity. As current literature is still very confusing regarding MC`s role in and around the tumor tissue, this in vitro study may help enlighten its interactions with tumor cells and trigger new explanatory in vitro/in vivo studies like, for example, delineating the killing

mediators, are discussed by us in detail somewhere (Özdemir, 2006).

1998; Özdemir, 2006; Ribatti, 2009; Aldinucci, 2010).

models.

**5. Conclusion** 

mechanisms of MCC.

necrotic killing in Daudi/Raji, which probably reflects pro-necrolytic characteristics of MCC.

Early cell death of LAK-sensitive/-resistant cells in our experiments detected from 2h indicated human MCC occurring in short-term coincubation. These results strongly suggest a possible contribution of a fast-acting secretory pathway via the exocytosis of pro-apoptotic granules of MCs to the human MCC. Accordingly, these findings verify the recent literature suggestive of MC-induced apoptosis in smooth muscle cell, cardiomyocytes and endothelial cells (Leskinen, 2003; Heikkilä, 2008).

The killing at various coincubation times in all LAK-resistant cells including patient cells was close to statistical significance partly due to an inadequate number of experiments. However, it was still noticeably important since these cells are known to be resistant to even LAK cells, and they are not essentially TNF-α sensitive cell. Thus our results also indicate a role for MC as a contributory effector cell in cellular immune surveillance of human innate immunity, resembling recently reported studies (Marshall, 2004; Özdemir, 2006).

Contrary to the findings of this in vitro study, MC availability in tissues and tumor stroma has still been controversial. The important point is here: increased tissue MCD could be primary and/or secondary since MCD is also found to be increased physiologically around healing tissue (Özdemir, 2006). MCs might be just as a reflection of generalized inflammatory reaction as well. For instance; the infiltration of MCs was thought to be a reflection of the host inflammatory response and is favorable prognostic factor in diffuse large B-cell lymphoma (Hedström, 2007). Similarly, MCs were believed to represent reactive cell types involve in the pathophysiology of the host reaction in lymphoma (Sharma, 1992). MCs also accumulate at sites of tumor growth in response to numerous chemoattractants or mediators. For example, CCL5/CXCR3 chemokines in lymphoma and tumor-derived stem cell factor and CD30 expressions lead to tumor growth and MCD in tumor tissues (Fischer, 2003). While evaluating the tumor-MC relationship, not only the MCD, but several other factors, such as the relationship of MCs with other stromal (fibroblasts, endothelial) and inflammatory cells should also be considered (Aldinucci, 2010). Hence it is important to find an answer to the following question: can the increased MCD be a result of tumor progression or a cause of tumor progression and a poor prognosis? Then the next question becomes, is the MC really an active player or an innocent passerby in a tumor stroma?

Increased MCD in the tumor stroma was assumed to be a stimulator of tumor progression through angiogenesis. As with other tumors; there are also conflicting results about the role of MCD and its relation to microvessel density (MVD) in lymphoma/leukemia (Özdemir, 2006). In addition, lymphoma progression is recently considered to be potentiated by at least two distinct angiogenic mechanisms. Autocrine stimulation of tumor cells via expression of vascular endothelial growth factor (VEGF) and VEGF receptors by lymphoma cells, as well as paracrine influences of proangiogenic tumor stroma on both local neovascularization and recruitment of circulating BM-derived progenitors (Ruan, 2009). Similarly, isolated AML blasts were shown to overexpress the VEGF/VEGFR–2 pathway, which can promote the growth of leukemic blasts in an autocrine and paracrine manner (Mesters, 2001). Yet, in some literature the following are hematological tumors in which MCD correlates with MVD: B-cell non-Hodgkin lymphoma, HL, follicular/angioimmunoblastic T-cell lymphoma, and B-cell chronic lymphocytic leukemia (Ribatti, 1998; Fukushima, 2001; Ribatti, 2009; Tripodo, 2010; Taskinen, 2010). And human tumors in which MCD is supposed to correlate with a poor prognosis include HL, diffuse large B-cell lymphoma, follicular and angioimmunoblastic T-cell lymphoma (Molin, 2002; Gratzinger, 2007; Ribatti, 2009; Taskinen, 2010;). Amazingly, in some studies, human tumors in which MCD is supposed to correlate with a good prognosis include HL, diffuse large B-cell lymphoma, and follicular lymphoma (Koster, 2005; Hedström, 2007; Rygoł, 2007;Ribatti, 2009;). Although it is hard to explain these conflicting results above and in the literature, they may be associated with different methodologies used in studies such as timing of biopsy (e.g. doing in early instead of late stage of tumor), the tumor type, as well as environmental factors surrounding that tumor (Özdemir, 2006). Only observing increased MCD in various tumors with good or bad prognosis on pathological specimens seems to be far behind to explain the real role of MCs. The net effect of MCs on tumor growth, therefore, is likely to be the result of multiple interactions between MC, tumor, and associated inflammatory cells with their signaling pathways, and adjacent stromal cells such as vascular endothelium and fibroblasts (Ribatti, 1998; Özdemir, 2006; Ribatti, 2009; Aldinucci, 2010).

Consequently, antiangiogenic strategies have recently become an important therapeutic modality for solid tumors. And MCs have been thought of as a new target for the adjuvant treatment of tumors through the selective inhibition of angiogenesis, tissue remodeling and tumor promoting molecules. Preliminary studies with cromolyn in mice mammary adenocarinoma and pancreatic cancer therapy was ineffective. Although some therapeutics like alemtuzumab were also given as an example of antiangiogenic treatment success, it is known as an anti-myeloid cell antiangiogenic agent used for the treatment of ovarian cancer, not an anti-MC agent (Ruan, 2009). Beside MCs and myeloid cells, Tie2-expressing monocytes and vascular leukocytes have recently been shown as new targets in the regulation of tumor associated angiogenesis (De Palma, 2007). In the age of targeted therapy, studies of the targeting MCs` role in cancer might have direct clinical consequences and should be further elucidated via the use of histopathological and complex biological models.

Since the aim of this study was basically to show MCC using human effector/target cells, the mechanisms and specificity of human MCC were not evaluated in this study. Our studies still continue studying MC-tumor cell relations and the characteristics of MCC on human pathological biopsy specimens. We hypothesize that human MCC may happen thru non-secretory pathway (cell-to-cell contact) of membranous TNF-α/Fas L, and secretory pathway via released mediators like soluble TNF-α, granzymes and chymase. Simply, MCC happens via pro-apoptotic/-necrolytic granules of MCs in the short term, as well as through its membranous components (non-secretory pathway of MCC) in the long term as shown in this study. Consistently, observing effector-target doublets on Wright-Giemsa slides of this study might suggest the importance of cell-to-cell contact in this process. The mechanisms with characteristics of human MCC against human tumor cells, as well as the role of MC mediators, are discussed by us in detail somewhere (Özdemir, 2006).

#### **5. Conclusion**

16 Advances in Cancer Therapy

necrotic killing in Daudi/Raji, which probably reflects pro-necrolytic characteristics of

Early cell death of LAK-sensitive/-resistant cells in our experiments detected from 2h indicated human MCC occurring in short-term coincubation. These results strongly suggest a possible contribution of a fast-acting secretory pathway via the exocytosis of pro-apoptotic granules of MCs to the human MCC. Accordingly, these findings verify the recent literature suggestive of MC-induced apoptosis in smooth muscle cell, cardiomyocytes and endothelial

The killing at various coincubation times in all LAK-resistant cells including patient cells was close to statistical significance partly due to an inadequate number of experiments. However, it was still noticeably important since these cells are known to be resistant to even LAK cells, and they are not essentially TNF-α sensitive cell. Thus our results also indicate a role for MC as a contributory effector cell in cellular immune surveillance of human innate

Contrary to the findings of this in vitro study, MC availability in tissues and tumor stroma has still been controversial. The important point is here: increased tissue MCD could be primary and/or secondary since MCD is also found to be increased physiologically around healing tissue (Özdemir, 2006). MCs might be just as a reflection of generalized inflammatory reaction as well. For instance; the infiltration of MCs was thought to be a reflection of the host inflammatory response and is favorable prognostic factor in diffuse large B-cell lymphoma (Hedström, 2007). Similarly, MCs were believed to represent reactive cell types involve in the pathophysiology of the host reaction in lymphoma (Sharma, 1992). MCs also accumulate at sites of tumor growth in response to numerous chemoattractants or mediators. For example, CCL5/CXCR3 chemokines in lymphoma and tumor-derived stem cell factor and CD30 expressions lead to tumor growth and MCD in tumor tissues (Fischer, 2003). While evaluating the tumor-MC relationship, not only the MCD, but several other factors, such as the relationship of MCs with other stromal (fibroblasts, endothelial) and inflammatory cells should also be considered (Aldinucci, 2010). Hence it is important to find an answer to the following question: can the increased MCD be a result of tumor progression or a cause of tumor progression and a poor prognosis? Then the next question becomes, is the MC really an active player or an innocent passerby in a tumor stroma? Increased MCD in the tumor stroma was assumed to be a stimulator of tumor progression through angiogenesis. As with other tumors; there are also conflicting results about the role of MCD and its relation to microvessel density (MVD) in lymphoma/leukemia (Özdemir, 2006). In addition, lymphoma progression is recently considered to be potentiated by at least two distinct angiogenic mechanisms. Autocrine stimulation of tumor cells via expression of vascular endothelial growth factor (VEGF) and VEGF receptors by lymphoma cells, as well as paracrine influences of proangiogenic tumor stroma on both local neovascularization and recruitment of circulating BM-derived progenitors (Ruan, 2009). Similarly, isolated AML blasts were shown to overexpress the VEGF/VEGFR–2 pathway, which can promote the growth of leukemic blasts in an autocrine and paracrine manner (Mesters, 2001). Yet, in some literature the following are hematological tumors in which MCD correlates with MVD: B-cell non-Hodgkin lymphoma, HL, follicular/angioimmunoblastic T-cell lymphoma, and B-cell chronic lymphocytic leukemia (Ribatti, 1998; Fukushima, 2001; Ribatti, 2009; Tripodo, 2010; Taskinen, 2010). And human tumors in which MCD is supposed to correlate with a poor prognosis include HL, diffuse large B-cell lymphoma, follicular and angioimmunoblastic T-cell lymphoma (Molin, 2002; Gratzinger, 2007; Ribatti, 2009;

immunity, resembling recently reported studies (Marshall, 2004; Özdemir, 2006).

MCC.

cells (Leskinen, 2003; Heikkilä, 2008).

Our findings in this study verify possible anti-tumor role of MC as a contributory effector cell to NK and other cytotoxic cells in immunosurveillance within human innate immunity. As current literature is still very confusing regarding MC`s role in and around the tumor tissue, this in vitro study may help enlighten its interactions with tumor cells and trigger new explanatory in vitro/in vivo studies like, for example, delineating the killing mechanisms of MCC.

May Mast Cells Have Any Effect in New Modalities of Cancer Treatment? 19

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Ribatti, D. & Crivellato, E. (2009). The controversial role of mast cells in tumor growth. *International Review of Cell and Molecular Biology*. Vol.275, pp. 89-131. Molin, D.; Edström, A.; Glimelius, I.; et al. (2002). Mast cell infiltration correlates with poor

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These conflicting reports might indicate that there seem to be other factors determining the relationship between MCD and tumor progression. The conflicting results may also depend on wide variations in timing, tumor types/stages, methodologies, and the chemotherapy application as well. Importantly, some studies are not able to reflect the direct effects of MCs on tumor biology because many patients receive adjuvant chemotherapy in the time of sampling. So, some of the effects can be accounted for by the therapeutic response to chemotherapy. Nonetheless, by only observing increased MCD in and around a tumor, making a correlation between good or bad prognosis and specimens seems inadequate to explain their real role.
