The Influences of MSC Infused via Different The Influences of MSC Infused via Different Ways and Different Time on Anti-tumor Ways and Different Time on Anti-tumor Activities of CIK/NK cells from Umbilical Activities of CIK/NK cells from Umbilical Cord Blood in K562 NOD/SCID MiceCord Blood in K562 NOD/SCID Mice

黎阳 吴燕峰 王潇娉 黄绍良 方建培 黄科 魏菁黎阳 吴燕峰 王潇娉 黄绍良 方建培 黄科 魏菁

中山大学孙逸仙纪念医院儿科中山大学孙逸仙纪念医院儿科 drliyang@126.comdrliyang@126.com

BackgroundBackgroundBone marrow mesenchymal stem cells Bone marrow mesenchymal stem cells (MSC) has a unique immunological (MSC) has a unique immunological characteristicscharacteristics ：：Low expression of class MHC moleculesⅠLow expression of class MHC moleculesⅠDo not express class MHC moleculesⅡDo not express class MHC moleculesⅡDid not express Fas ligand and co-stimulatory Did not express Fas ligand and co-stimulatory moleculesmolecules

Therefore, MSC is survival possible long-Therefore, MSC is survival possible long-term in the host body, even can be term in the host body, even can be survival long-term in heterogeneity host.survival long-term in heterogeneity host.

BackgroundBackground

Cytokine-induced killer(CIK) cellsCytokine-induced killer(CIK) cells are heterogeneous are heterogeneous

group of cells that obtained from co-cultured mononuclear group of cells that obtained from co-cultured mononuclear

cells of the human peripheral blood, cord blood or bone cells of the human peripheral blood, cord blood or bone

marrow with stimulation of variety cytokines such as IL-2, marrow with stimulation of variety cytokines such as IL-2,

IFN-γIFN-γ ，， Anti-CD3 in vitro after a period of time.Anti-CD3 in vitro after a period of time.

CIK cells expressing CD3 and CD56. CIK cells expressing CD3 and CD56.

BackgroundBackground Studies have showed that MSC has a Studies have showed that MSC has a

negative regulatory role, whether or not its negative regulatory role, whether or not its

can effect the anti-tumor activity of can effect the anti-tumor activity of

subsequent infused killer cells in vivo is subsequent infused killer cells in vivo is

worth being further studied.worth being further studied.

BackgroundBackground

Because CD3Because CD3++CD56CD56++ CIK cells derived CIK cells derived from CD3from CD3++CD56CD56-- T cells, MSC may also T cells, MSC may also inhibit anti-tumor activity of CD3inhibit anti-tumor activity of CD3++CD56CD56++ CIK cells.CIK cells.

ObjectivesObjectives

To explore the influences of MSC To explore the influences of MSC

infused via different ways and at infused via different ways and at

different time on anti-tumor activities of different time on anti-tumor activities of

CIK/NK cells from umbilical cord blood CIK/NK cells from umbilical cord blood

in K562 NOD/SCID mice.in K562 NOD/SCID mice.

Experimental prinipleExperimental priniple ：：

MSC CIK/NK cells

Inhibition

infusion

infusion

K562 NOD/SCID mice

Shorten survival Increase in tumor burden

Experimental groups ：

NOD/SCID mice

MSC CIK/NK

Group Aat the same

time

(caudal vein) (caudal vein)

Group B

Delayed 48h

MSC CIK/NK(caudal vein) (caudal vein)

Group D

Group C

MSC CIK/NK (marrow cavity) (caudal vein)

at the same time

Delayed 48h

MSC CIK/NK (marrow cavity) (caudal vein)

Group ECIK/NK

(caudal vein)

Group FDelayed 48 hours

CIK/NK(caudal vein)

Group GK562 cells

(inoculated alone)

K562After 12h After 12h

Detection of indicators: Detection of indicators: Calculate survival curve of each group Calculate survival curve of each group

NOD/SCID tumor-bearing miceNOD/SCID tumor-bearing mice By morphology, flow cytometry, and By morphology, flow cytometry, and

histopathology,to detect the tumor cell histopathology,to detect the tumor cell loads in peripheral blood, bone marrow, loads in peripheral blood, bone marrow, liver, spleen ,lungs and other organs.liver, spleen ,lungs and other organs.

Survival time and body weight change in various Survival time and body weight change in various group of NOD/SCID mice(x±s,n=8) group of NOD/SCID mice(x±s,n=8)

GroupGroup the former body weight the former body weight Near-death or 60d in body weightNear-death or 60d in body weight Survival timeSurvival time

（（ gg ） （ ） （ gg ） （ ） （ dd））A 23.5±1.3 18.0±0.7A 23.5±1.3 18.0±0.7▲▲ 36.1±9.9 36.1±9.9■■

B 22.3±1.9 18.1±1.8B 22.3±1.9 18.1±1.8▲▲ 35.9±10.1 35.9±10.1■■

C 23.8±1.7 21.3±2.5 49.8±8.9 C 23.8±1.7 21.3±2.5 49.8±8.9 D 23.9±0.9 20.3±1.6 47.9±9.0D 23.9±0.9 20.3±1.6 47.9±9.0E 22.5±1.8 20.5±2.2 49.8±11.2E 22.5±1.8 20.5±2.2 49.8±11.2F 23.0±0.7 20.4±0.5 49.1±8.9F 23.0±0.7 20.4±0.5 49.1±8.9G 22.8±1.7 17.9±1.9G 22.8±1.7 17.9±1.9▲▲ 29.4±3.4 29.4±3.4■■

Note: ▲to compare C, D, E, F set of data,there was statistically significant difference (P <0.05);         ■to compare the C, D, E, F set of data,ther was statistically significant difference (P <0.05).

Kaplan-Meier survival curve in various group of Kaplan-Meier survival curve in various group of NOD/SCID mice NOD/SCID mice

1. the average survival time of mice in groups A and B was significantly shorter than groups C, D, E and F.2. the average survival time of mice was no significant difference between groups A and B.3. the average survival time of mice was no significant difference between groups C and D.4. the average survival time of mice was no significant difference among groups A and B and group G.

Proportion of tumor cells in the liver, spleen, lung tissue homogenate in Proportion of tumor cells in the liver, spleen, lung tissue homogenate in each group miceeach group mice

Group liver Group liver spleenspleen lunglungA 8.5%A 8.5% ～～ 19% 0%19% 0% ～～ 4% 13%4% 13% ～～25%25%B 4.5%B 4.5% ～～ 15% 0%15% 0% ～～ 3.5% 7%3.5% 7% ～～17.5%17.5%C 3%C 3% ～～ 9.5% 0%9.5% 0% ～～ 2% 3.5%2% 3.5% ～～10%10%D 4%D 4% ～～ 11.5% 0%11.5% 0% ～～ 0.5% 4%0.5% 4% ～～11.5% 11.5% E 3.5%E 3.5% ～～ 10.5% 0% 4.5%10.5% 0% 4.5% ～～12% 12% F 5%F 5% ～～ 8% 0%8% 0% ～～ 1% 5%1% 5% ～～11%11%G 15%G 15% ～～ 20% 1%20% 1% ～～ 5% 20%5% 20% ～～27% 27%

Liver, spleen, lung tissue homogenate smears: the proportion of tumor cells in mice of group A and B is higher than that of group C, D, E and F; the proportion of tumor cells in mice of group A is near to group B;the proportion of tumor cells in mice of group C is near to group D;the proportion of tumor cells in mice of group A and B is near to group G.

CD33 expression level in different parts of each CD33 expression level in different parts of each group (x±sgroup (x±s ，， n=8)(%)n=8)(%)

Group Group PB BM PB BM liverliver spleenspleen lunglung

A 1.59±0.6 1.43±0.7 4.86±1.6A 1.59±0.6 1.43±0.7 4.86±1.6▲ ▲ 3.30±1.1 3.30±1.1■■ 11.45±1.6 11.45±1.6●●

B 1.28±0.9 1.20±0.9 3.34±1.6B 1.28±0.9 1.20±0.9 3.34±1.6◆◆ 1.83±1.2 7.47±2.6 1.83±1.2 7.47±2.6●●

C 0.98±0.6 0.60±0.4 1.95±0.7C 0.98±0.6 0.60±0.4 1.95±0.7▲▲ 0.59±0.4 6.10±1.0 0.59±0.4 6.10±1.0●●

D 0.43±0.5 0.74±0.4 2.28±1.1D 0.43±0.5 0.74±0.4 2.28±1.1★★ 0.64±1.0 2.83±1.8 0.64±1.0 2.83±1.8★★

E 0.74±0.3 0.77±0.5 2.31±1.0E 0.74±0.3 0.77±0.5 2.31±1.0▲▲ 0.65±1.1 1.25±1.1 0.65±1.1 1.25±1.1

F 0.44±0.2 0.51±0.4 1.82±1.3F 0.44±0.2 0.51±0.4 1.82±1.3★★ 0.59±0.5 1.25±1.1 0.59±0.5 1.25±1.1▼▼

G 1.60±0.7 1.52±0.8 5.36±1.5G 1.60±0.7 1.52±0.8 5.36±1.5▲▲ 3.31±1.1 3.31±1.1■■ 11.89±1.5 11.89±1.5●●

CD33 expression in lung and liver tissue homogenate were significantly higher than those in peripheral blood and bone marrow in each group mice.(P＜ 0.05 ）

Visible liver tumor in Group A mice

Pathological section of liver tumor in group A mince (HE staining, up 40 ×; down 400 ×)

Pathological section of lung tumor in group B mince

(HE staining, up 40 ×; under 400 ×)

Visible lung tumor in Group B mice

ConclusionsConclusions

conclusion conclusion In vivo ,MSC may significantly inhibit In vivo ,MSC may significantly inhibit

anti-tumor activity of killer cell with the anti-tumor activity of killer cell with the same way infusion. same way infusion.

Even if infused MSC 2 days in advance, Even if infused MSC 2 days in advance,

this inhibitory effect still exists.this inhibitory effect still exists.

conclusion conclusion Through the tumor-bearing animal model Through the tumor-bearing animal model

study,it revealed that pre-entered MSC in vivo study,it revealed that pre-entered MSC in vivo will weaken GVL effect of the CIK / NK cells.will weaken GVL effect of the CIK / NK cells.

It prompts that by using MSC combine with It prompts that by using MSC combine with HSCT,clearance of MRD and the GVL effect HSCT,clearance of MRD and the GVL effect may be detrimental to patients with may be detrimental to patients with hematologic malignancies,and may affect hematologic malignancies,and may affect anti-tumor activity of the latter infused CIK anti-tumor activity of the latter infused CIK cells, NK cells or CTL, thereby increase the cells, NK cells or CTL, thereby increase the risk of tumor recurrence.risk of tumor recurrence.