**3. Results**

10 Advances in Cancer Therapy

An average of 10,000 total events and 3,000 target cells were collected per sample. The gating for the target cells was based on the target-alone analysis and kept constant throughout all tubes to avoid exaggeration of the counts due to apoptotic body contamination. Cytotoxicity calculations were based on viable populations in target-alone (control) and co-culture (coincubated) tube analysis results. Viable target-cell percentage was determined, and calculations were based on the control-tube (target alone) values. Apoptotic and/or necrotic death happening in control tubes were identified as spontaneous, but those killed in coincubated tubes were identified as cytotoxic killing. We expressed the

As mentioned, we defined MCC occurring in co-culture tube (coincubated sample) as percent (%, PC) cytotoxicity. And cytotoxicity calculations were based on viable

Control-viable cell% – Coincubation-viable cell% Percent Cytotoxicity PC = Control-viable cell%

Our in vitro experiments were repeated several times (n=27) with a few sets of MC colonies. Samples were tested in duplicates for reproducibility and reliability (mean coefficients of

A standard assay was performed as previously described (Özdemir, 2007). Briefly, the assay is a standard 4h CRA using target cells that have been prelabeled with 100 μCi 51Cr (Perkin Elmer, Boston, MA) for 1h. Several concentrations of MCs were added to a fixed number of target cells (5,000) in a round bottom microtiter plate to a total volume of 0.2 ml. Following the 4h incubation, 0.1 ml of the supernatant was carefully harvested and counted on a scintillation counter (Packard, Downers Grove, IL). Maximum release was determined from wells including target cells and 10% sodium dodecyl sulfate in the medium. The PC was calculated using the following equation: (E–S)/(M–S)x100, where E is the experimental counts/minute, S is the spontaneous counts/minute and M is the maximum counts/minute. In the end, when we used some sets of our experimental data in comparing FCM cytotoxicity assays with CRA results, there was a significant correlation for PC (n=58; r=

At the end of the coincubation period, the sample was gently mixed and 10µl was used to prepare slides at room temperature without cytospinning. The slides were then stained with Wright/Giemsa. Conjugate formations between some tumor cells and MCs were seen

Unless otherwise specified, all data are presented as means±SEM. The significance of differences between spontaneous and cytotoxic kill was determined using the paired

 

populations in target-alone (control) and co-culture tube analysis results.

variation (CV) of 1.1% (95% CI, 0.8- 1.7%); r2=0.93, and p<0.001).

**2.11 Comparison with chromium- 51Cr release assay (CRA)** 

0.95; P<0.001), similar to our earlier studies (Özdemir, 2007, 2011).

**2.12 Cell staining with Wright/Giemsa for light microscopy** 

(Fig.1A1-A4).

**2.13 Statistical analysis** 

**2.9 FCM data analysis** 

MCC as percentage based cytotoxicity:

**2.10 Percentage based cytotoxicity (%, PC)** 

This study demonstrated MCC against tumor cells by using human cells instead of murine effector/target cells. In vitro MCC against various tumor cells at different E:T ratios (1:1-5:1) was measured by FCM-MCMCA and DIOC18 methods on various periods (Table 2A-D).


The death/killings (percentage based cytotoxicity) in this table reflect the death at a 5:1 ratio in this study. Spontaneous kill reflects the death in target or effector alone tubes. Cytotoxic death shows the target cell death by only mast cell- mediated cytotoxicity in coincubation sample. The percentage based cytotoxicity was calculated according to the given formula in the methods. All values are given as mean±SEM. P values reflect the significance of differences between spontaneous and mast cell mediated cytotoxic killing. N denotes the number of experiments were repeated in each series. NS means not significant.

Table 2A. In vitro human mast cell -mediated cytotoxicity measured by DIOC18 method against different human tumor cells is shown.

May Mast Cells Have Any Effect in New Modalities of Cancer Treatment? 13

**Percentage Based Human Mast Cell- Mediated Cytotoxicity (%, PC) Cell characteristics %, PC on Coincubation Times LAK-sensitive Death Type 2h 12h 18h 24h 48h** 

> 2±1 3±1 0±1 (3) NS

4±1 4±1 30±1 (3) **0.030** 

5±1 7±1 19±1 (3) **0.044** 

**LAK-resistant Death Type 2h 12h 18h 24h 48h** 

5±1 2±1 6±1 (3) NS

3±1 1±0 4±1 (3) NS

3±1 5±1 5±1 (3) NS

3±1 2±1 5±1 (6) NS

The death/killings (percentage based cytotoxicity) in this table reflect the death at a 2:1 ratio in this study. Spontaneous kill reflects the death in target or effector alone tubes. Cytotoxic death shows the target cell death by only mast cell-mediated cytotoxicity in the coincubation sample. The percentage based cytotoxicity was calculated should be given formula. All values are given as mean±SEM. P values reflect the significance of differences between spontaneous and mast cell mediated cytotoxic killing. N denotes the number of experiments were repeated in each series. NS means not significant. Table 2C. In vitro human mast cell -mediated cytotoxicity (%, PC) against different human

4±1 4±1 6±1 (9) NS

3 ± 2 3 ± 1 57± 2 (9) **0.001** 

2 ± 1 5 ± 1 26 ± 1 (9) **0.016** 

3±1 5±1 15±2 (6) NS

5±1 6±1 14±2 (5) NS

6±1 7±1 13±1 (6) NS

2±1 6±1 15±1 (6) NS

3±1 5±1 6±1 (9) NS

3 ± 2 4 ± 1 59± 2 (9) **0.001**

2 ± 1 7 ± 1 46 ± 1 (9) **0.006**

4±1 5±1 17±2 (6) NS

4±1 5±1 18±2 (5) NS

3±1 8±1 16±1 (6) NS

4±1 8±1 18±1 (6) NS

5±1 6±1 14±1 (9) NS

4 ± 2 5 ± 2 63 ± 3 (9) **0.001** 

4 ± 1 9 ± 3 67± 3 (9) **0.001** 

6±1 7±1 20±2 (6) NS

6±1 8±1 22±4 (5) NS

5±1 9±2 28±3 (6) **0.034** 

6±1 10±1 20±2 (6) NS

7±1 5±1 16±2 (9) NS

4 ± 2 8 ± 2 65 ± 3 (9) **0.001**

4 ± 1 10 ± 3 75± 3 (9) **0.001** 

8±1 7±1 22±2 (6) NS

6±1 8±1 25±4 (5) **0.039** 

5±1 11±2 36±3 (6) **0.019** 

6±1 13±1 21±2 (6) NS

Spontaneous Spontaneous Cytotoxic N= P=

Spontaneous Spontaneous Cytotoxic N= P=

Spontaneous Spontaneous Cytotoxic N= P=

Spontaneous Spontaneous Cytotoxic N= P=

Spontaneous Spontaneous Cytotoxic N= P=

Spontaneous Spontaneous Cytotoxic N= P=

Spontaneous Spontaneous Cytotoxic N= P=

tumor cells measured by both cytotoxicity assays is shown at various periods.

Effector (**Mast**) control/alone Target (**K562**) control Coincubation (2:1)

Effector (**Mast**) control/alone Target (**DAMI**) control/alone

Coincubation (2:1)

Effector control Target (**Meg-01**) control Coincubation (2:1)

Effector control Target (**HL-60**) control Coincubation (2:1)

Effector control

Target (**Patients`**) control Coincubation (2:1)

Effector control Target **(Daudi**) control Coincubation (2:1)

Effector control Target (**Raji**) control Coincubation (2:1)


The death/killings (percentage based cytotoxicity) in this table reflect the death at 1:1, 2:1, 4:1 ratios in this study. Spontaneous kill reflects the death in target or effector alone tubes. Cytotoxic death shows the target cell death by only mast cell-mediated cytotoxicity in the coincubation sample. The percentage based cytotoxicity was calculated according to the given formula in the methods. All values are given as mean±SEM. P values reflect the significance of differences between spontaneous and mast cell mediated cytotoxic killing. N denotes the number of experiments were repeated in each series. NS means not significant.

Table 2B. In vitro human mast cell-mediated cytotoxicity % (percentage based killing) measured by FCM-MCMCA against human LAK-sensitive tumor cells at different ratios and time periods (2h- 24h) are shown.

**Percentage Based Human Mast Cell- Mediated Cytotoxicity (%, PC)** 

**LAK-sensitive Death Type 2h 12h 24h** 

Spontaneous Spontaneous Cytotoxic N= P=

Spontaneous Spontaneous Cytotoxic N= P=

Spontaneous Spontaneous Cytotoxic N= P=

Spontaneous Spontaneous Cytotoxic N= P=

Spontaneous Spontaneous Cytotoxic N= P=

Spontaneous Spontaneous Cytotoxic N= P=

The death/killings (percentage based cytotoxicity) in this table reflect the death at 1:1, 2:1, 4:1 ratios in this study. Spontaneous kill reflects the death in target or effector alone tubes. Cytotoxic death shows the target cell death by only mast cell-mediated cytotoxicity in the coincubation sample. The percentage based cytotoxicity was calculated according to the given formula in the methods. All values are given as mean±SEM. P values reflect the significance of differences between spontaneous and mast cell mediated cytotoxic killing. N denotes the number of experiments were repeated in each series. NS

Table 2B. In vitro human mast cell-mediated cytotoxicity % (percentage based killing) measured by FCM-MCMCA against human LAK-sensitive tumor cells at different ratios

Effector (**Mast**) control Target (**Daudi**) control Coincubation (1:1)

Effector control/alone Target (Daudi) control/alone

Effector control /alone Target (**Raji**) control /alone

Coincubation (1:1)

Effector alone Target (Raji) alone Coincubation (2:1)

Effector alone Target (Raji) alone Coincubation (4:1)

means not significant.

and time periods (2h- 24h) are shown.

Coincubation (2:1)

Effector control Target (Daudi) control Coincubation (4:1)

**Cell characteristics %, PC on Coincubation Times** 

3 ± 1 2 ± 1 21 ± 2 (9) **0.048** 

4±1 4±1 31±1 (3) **0.039** 

4±1 2±1 36±1 (9) **0.030** 

2±1 3±1 13±1 (9) **0.049** 

5±1 7±1 19±1 (3) **0.044** 

4±1 3±1 19±1 (9) **0.040** 

4 ± 1 3 ± 1 49 ± 2 (9) **0.009** 

3 ± 2 3 ± 1 57± 2 (9) **0.001** 

3 ± 2 4 ± 1 59± 2 (9) **0.001** 

3 ± 1 4 ± 1 16 ± 1 (9) **0.046** 

3 ± 1 5 ± 1 26 ± 1 (9) **0.016** 

6 ± 1 7 ± 1 30 ± 1 (9) **0.014** 

5 ± 2 6 ± 2 53 ± 3 (9) **0.005** 

4 ± 2 5 ± 2 63 ± 3 (9) **0.001** 

4 ± 2 6 ± 2 67 ± 3 (9) **0.001** 

6 ± 1 8 ± 3 47± 3 (9) **0.008** 

4 ± 1 9 ± 3 67± 3 (9) **0.001** 

5 ± 1 8 ± 3 69± 3 (9) **0.001** 


The death/killings (percentage based cytotoxicity) in this table reflect the death at a 2:1 ratio in this study. Spontaneous kill reflects the death in target or effector alone tubes. Cytotoxic death shows the target cell death by only mast cell-mediated cytotoxicity in the coincubation sample. The percentage based cytotoxicity was calculated should be given formula. All values are given as mean±SEM. P values reflect the significance of differences between spontaneous and mast cell mediated cytotoxic killing. N denotes the number of experiments were repeated in each series. NS means not significant.

Table 2C. In vitro human mast cell -mediated cytotoxicity (%, PC) against different human tumor cells measured by both cytotoxicity assays is shown at various periods.

May Mast Cells Have Any Effect in New Modalities of Cancer Treatment? 15

was close to statistical significance, partly due to an inadequate number of experiments (Table 2A,C). In overall; Daudi/Raji/Meg-01 and HL-60 cell deaths were found to be statistically significant at different periods in long term, compared to spontaneous (control) killing (Table 2A-C). Thus, these findings reveal human MC`s cytotoxic capacity against

Briefly, interestingly, MCs did not seem to be very effective against some type of human NK-/LAK-sensitive cells such as K562; but LAK-resistant cells including Meg-01/HL-

The ability to study MCC in longer coincubation times (≤48h), in addition to shorter coincubation times (2h-18h), is another advantage of our established FCM methods in this study. This appears to be a drawback for CRA as well as other methods utilizing dyes such as fluorochromes (PKH-26) due to increased risk in release of 51Cr or dye leakage, which results in staining of other populations. Nevertheless, our applications are potentially important for studying certain apoptotic pathways that take longer to become operational, such as membranous TNF-α-induced apoptosis, which is believed to be one of most important components of MCC. Membranous TNF-α has been shown to kill WEHI-164/L929/Raji cells in 24h assays (Henderson, 1981; Ghiara, 1985; Richards 1988; Heikkilä,

In this study, our results were found to be reproducible and reliable when experiments repeated (mean coefficients of variation (CV) of 1.1%). Incremental increase in MCC detected at different ratios/times shows reproducibility and reliability of our methods (Table 2A-C). When our flow cytometric data compared with CRA results, there was a significant correlation (n=104, r=0.82, p<0.001), similar to our earlier studies (Özdemir, 2003,

In addition, Wright-Giemsa slides showed conjugate formations between MC and some tumor cells (MC-target cell doublets), indicating possible initial steps of human MCC, perhaps via cell-to-cell contact thru their membranous components such as membranous

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

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

human LAK-resistant/-sensitive cells.

2008; Özdemir, 2003, 2006).

TNF-α/Fas Ligand (Fig.1A1-4).

recently published (Özdemir, 2003, 2007, 2011).

2007, 2011).

**4. Discussion** 

60/DAMI/patients' were killed somewhat effectively by MCC.


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 sample.

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

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 MCC.

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 too.

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' was close to statistical significance, partly due to an inadequate number of experiments (Table 2A,C). In overall; Daudi/Raji/Meg-01 and HL-60 cell deaths were found to be statistically significant at different periods in long term, compared to spontaneous (control) killing (Table 2A-C). Thus, these findings reveal human MC`s cytotoxic capacity against human LAK-resistant/-sensitive cells.

Briefly, interestingly, MCs did not seem to be very effective against some type of human NK-/LAK-sensitive cells such as K562; but LAK-resistant cells including Meg-01/HL-60/DAMI/patients' were killed somewhat effectively by MCC.

The ability to study MCC in longer coincubation times (≤48h), in addition to shorter coincubation times (2h-18h), is another advantage of our established FCM methods in this study. This appears to be a drawback for CRA as well as other methods utilizing dyes such as fluorochromes (PKH-26) due to increased risk in release of 51Cr or dye leakage, which results in staining of other populations. Nevertheless, our applications are potentially important for studying certain apoptotic pathways that take longer to become operational, such as membranous TNF-α-induced apoptosis, which is believed to be one of most important components of MCC. Membranous TNF-α has been shown to kill WEHI-164/L929/Raji cells in 24h assays (Henderson, 1981; Ghiara, 1985; Richards 1988; Heikkilä, 2008; Özdemir, 2003, 2006).

In this study, our results were found to be reproducible and reliable when experiments repeated (mean coefficients of variation (CV) of 1.1%). Incremental increase in MCC detected at different ratios/times shows reproducibility and reliability of our methods (Table 2A-C). When our flow cytometric data compared with CRA results, there was a significant correlation (n=104, r=0.82, p<0.001), similar to our earlier studies (Özdemir, 2003, 2007, 2011).

In addition, Wright-Giemsa slides showed conjugate formations between MC and some tumor cells (MC-target cell doublets), indicating possible initial steps of human MCC, perhaps via cell-to-cell contact thru their membranous components such as membranous TNF-α/Fas Ligand (Fig.1A1-4).
