4.3 Effect of cell-cell interaction on CML chemoprotection
4.3.1 Cell-cell interaction with stromal cells for
To understand stroma mediated chemoresistance, first, K562 cells were co-cultured with MSC layer and treated with optimal concentration of IM (10µM) as determined earlier to induce apoptosis. After treatment for 48hours with IM, the adherent and suspension fractions of K562 in the co-culture system were separated and percentage of live cells was analyzed through Annexin-V/PI staining. A significantly high percentage of live cells were seen in stroma adherent fraction (AD-K562) compared to suspension fraction (SUS-K562) of co-cultured K562 cells during IM treatment (Figure 4.18).
Figure 4.18: Co-culture of K562 cells with MSC. Representative microscopic images of K562 CML cells cultured in suspension (left) or in co-culture with stromal cells (right) in presence of IM. (B) K562 cells co- cultured with MSC were treated with IM and the cells were separated into adherent (AD-K562) and
CML cells CML-stromal co-culture
50 60 70 80 90 100
SUS-K562 AD-K562
% Live cells
*
A B
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72
suspension (SUS-K562) fractions. The percentage of live cells was analyzed by Annexin-V/ PI staining through flow cytometry. A significantly high percentage of live cells were seen in AD-K562 compared to the SUS-K562 cells. Values are mean+SE, n=3; * p<0.05.
Moreover, the apoptosis percentage during IM treatment in SUS-K562 cells was similar to K562 cultured alone without MSC layer whereas it was significantly lower in AD- K562 cells (Figure 4.19). This suggests that cell-cell interaction protects K562 cells from chemo-drug induced cell death.
Figure4.19: Cell-cell interaction reduced IM induced apoptosis in K562 cells.K562 cells were co-cultured with stromal cells and treated with IM for 48 hours. The stroma adherent (AD-K562) and suspension fractions (SUS-K562) were separated and apoptosis was determined by Annexin-V/PI staining and compared with IM treated K562 cells grown without stromal layer (K562-CON). A significant reduction in the apoptosis percentage of IM treated AD-K562 was observed but the apoptosis in SUS-K562 was similar to that seen in K562-CON cells. (A) Pictorial representation of experimental set-up to analyze apoptosis in adherent and suspension cells. (B) Representative flow cytometric plots showing Annexin-V/PI staining in different cell fractions. (C) Graph representing the apoptosis percentage (An-V+PI- and An-V+PI+) of cell fractions after IM treatment. Values are mean+SD, n=5-6; ** p<0.005, *** p<0.0005.
To find out whether apoptotic pathway was activated in the IM treated AD-K562 and K562-CON cells, active-caspase 3 (active Cas-3) levels in the different cell fractions were detected. There was a significantly high percentage of active Cas-3 positive cells in IM treated K562-CON cells but it was significantly less in AD-K562 cell fraction (Figure 4.20) confirming the above data that cell-cell interaction indeed protects K562 cells from IM induced cell death.
SUS-K562 AD-K562 K562-CON
Annexin-V
PI
A
B
C
0 10 20 30 40 50
K562-CON SUS-K562 AD-K562
% Apoptotic cells
IM treated
***
**
4.0 RESULTS
73
Figure 4.20: Active caspase-3 staining in K562 cells.K562 cells were grown in co-culture with the stromal cells for 48hrs and stroma adherent (AD-K562) and control (K562-CON) cells were treated without or with IM for 48 hours. The AD-K56) and K562-CON cells were harvested and stained with fluorescently conjugated anti-active caspase-3 antibody. The cells were analyzed by flow cytometry and the percentage positive cells were calculated by gating against isotype stained control cells. (A) A significantly high percentage of active caspase-3 positive cells were seen in IM treated control cells (K562-CON+IM) compared to IM treatedAD-K562 cells (AD-K562+IM). Values are mean+SD, n=3 independent experiments. (B) Representative flow cytometric dot plot showing cells gated for active caspase-3 positive cells; ** p<0.005, *** p<0.0005.
During co-culture, as discussed before, K562 cells were present as both stroma adherent and non-adherent suspension cells. Hence, it might be possible in co-culture system that IM availability to the adherent K562 might be less due to the interference of suspension cells, because of which reduced cell death was observed in stroma adherent K562 cells.
To test this, initially, K562 cells were co-cultured with MSC layer to establish adherent and suspension fraction for 48hrs. After this, the suspension fraction was transferred to a new MSC layer and the adherent fraction was left undisturbed. IM was added to K562 cells present only as adherent cells or as suspension cells (Figure 4.21 A). Analysis of cell death percentage showed that apoptosis in AD-K562 cells was significantly less compared to SUS-K562 cells (Figure 4.21 B, C) confirming the above data that cell-cell interaction of K562 cells with stromal layer protects K562 cells from IM induced cell death irrespective of the drug availability.
Active caspase-3
FSC
K562-CON AD-K562
-IM+IM
0 10 20 30 40
K562-CON AD-K562 K562- CON+IM AD- K562+IM
Active caspase-3 +ve cells (%)
**
***
***
A B
4.0 RESULTS
74
Figure 4.21: Adherent K562 cells were chemoprotected. (A) Pictorial representation of experimental set- up. K562 cells were cultured in suspension or in co-culture with stromal cells. Only the suspension cells were transferred to a new stromal layer and adherent cells were retained on the same stromal layer. The control and both co-cultured cell fractions were treated with IM for 48 hours and apoptosis was determined by An-V/PI staining. (B) Representative flow cytometric plots showing An-V/PI staining. (C) A significant reduction in the apoptosis of stroma adherent K562 cells (AD-K562) was observed compared to the suspension fraction (SUS-K562). Values are mean+SE, n=3 independent experiments; * p<0.05.