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MUC16 provides immune protection by inhibiting synapse formation between NK and ovarian tumor cells.

Gubbels JA, Felder M, Horibata S, Belisle JA, Kapur A, Holden H, Petrie S, Migneault M, Rancourt C, Connor JP, Patankar MS - Mol. Cancer (2010)

Bottom Line: Our results indicate that MUC16-mediated inhibition of immune synapse formation is an effective mechanism employed by ovarian tumors to evade immune recognition.The NK cell leukemia cell line (NKL), which does not express KIRs but are positive for DNAM-1 and NKG2D, also conjugated and lysed MUC16-knockdown cells more efficiently than MUC16 expressing controls.The higher csMUC16 levels on the NKL resistant tumor cells correlated with more protection from lysis as compared to target cells that were never exposed to the effectors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Obstetrics and Gynecology, University of Wisconsin-Madison, Madison, WI, USA.

ABSTRACT

Background: Cancer cells utilize a variety of mechanisms to evade immune detection and attack. Effective immune detection largely relies on the formation of an immune synapse which requires close contact between immune cells and their targets. Here, we show that MUC16, a heavily glycosylated 3-5 million Da mucin expressed on the surface of ovarian tumor cells, inhibits the formation of immune synapses between NK cells and ovarian tumor targets. Our results indicate that MUC16-mediated inhibition of immune synapse formation is an effective mechanism employed by ovarian tumors to evade immune recognition.

Results: Expression of low levels of MUC16 strongly correlated with an increased number of conjugates and activating immune synapses between ovarian tumor cells and primary naïve NK cells. MUC16-knockdown ovarian tumor cells were more susceptible to lysis by primary NK cells than MUC16 expressing controls. This increased lysis was not due to differences in the expression levels of the ligands for the activating receptors DNAM-1 and NKG2D. The NK cell leukemia cell line (NKL), which does not express KIRs but are positive for DNAM-1 and NKG2D, also conjugated and lysed MUC16-knockdown cells more efficiently than MUC16 expressing controls. Tumor cells that survived the NKL challenge expressed higher levels of MUC16 indicating selective lysis of MUC16(low) targets. The higher csMUC16 levels on the NKL resistant tumor cells correlated with more protection from lysis as compared to target cells that were never exposed to the effectors.

Conclusion: MUC16, a carrier of the tumor marker CA125, has previously been shown to facilitate ovarian tumor metastasis and inhibits NK cell mediated lysis of tumor targets. Our data now demonstrates that MUC16 expressing ovarian cancer cells are protected from recognition by NK cells. The immune protection provided by MUC16 may lead to selective survival of ovarian cancer cells that are more efficient in metastasizing within the peritoneal cavity and also at overcoming anti-tumor innate immune responses.

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csMUC16 shields ovarian cancer cells from NKL mediated lysis. A. NKL cells were added to confluent cultures of csMUC16neg-OVC or csMUC16pos-OVC cells at a 1:1 effector:target ratio. After incubation for A, 24 h and B, 48 h the number of adherent colonies and adherent cells surviving the NKL challenge were counted by microscopic examination of five random fields. This experiment was repeated in triplicate and mean values (n = 15 fields) and standard deviation are shown.
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Figure 8: csMUC16 shields ovarian cancer cells from NKL mediated lysis. A. NKL cells were added to confluent cultures of csMUC16neg-OVC or csMUC16pos-OVC cells at a 1:1 effector:target ratio. After incubation for A, 24 h and B, 48 h the number of adherent colonies and adherent cells surviving the NKL challenge were counted by microscopic examination of five random fields. This experiment was repeated in triplicate and mean values (n = 15 fields) and standard deviation are shown.

Mentions: The ability of NKL cells to selectively lyse csMUC16neg-OVC and csMUC16pos-OVC was further evaluated in co-culture experiments. An additional MUC16-knockdown clone (csMUC16neg-OVC-A) was also used in these experiments. This protocol for the generation of this additional cell line has been reported [37]. The three sublines were plated at equal densities in separate wells. After cells were 70% confluent, NKL cells were introduced in the cultures and plates were incubated for 24 h (Figure 8A) or 48 h (Figure 8B). At the end of the incubation, NKL cells and lysed non-adherent ovarian tumor cells were removed and the remaining adherent colonies and individual cells were manually counted. Very few adherent colonies and cells per observed field remained in the csMUC16neg-OVC/NKL and csMUC16neg-OVC-A/NKL co-cultures (Figure 8). However, 3-4-fold more colonies and cells/field were detected in the csMUC16pos-OVC/NKL co-cultures (Figure 8).


MUC16 provides immune protection by inhibiting synapse formation between NK and ovarian tumor cells.

Gubbels JA, Felder M, Horibata S, Belisle JA, Kapur A, Holden H, Petrie S, Migneault M, Rancourt C, Connor JP, Patankar MS - Mol. Cancer (2010)

csMUC16 shields ovarian cancer cells from NKL mediated lysis. A. NKL cells were added to confluent cultures of csMUC16neg-OVC or csMUC16pos-OVC cells at a 1:1 effector:target ratio. After incubation for A, 24 h and B, 48 h the number of adherent colonies and adherent cells surviving the NKL challenge were counted by microscopic examination of five random fields. This experiment was repeated in triplicate and mean values (n = 15 fields) and standard deviation are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2818693&req=5

Figure 8: csMUC16 shields ovarian cancer cells from NKL mediated lysis. A. NKL cells were added to confluent cultures of csMUC16neg-OVC or csMUC16pos-OVC cells at a 1:1 effector:target ratio. After incubation for A, 24 h and B, 48 h the number of adherent colonies and adherent cells surviving the NKL challenge were counted by microscopic examination of five random fields. This experiment was repeated in triplicate and mean values (n = 15 fields) and standard deviation are shown.
Mentions: The ability of NKL cells to selectively lyse csMUC16neg-OVC and csMUC16pos-OVC was further evaluated in co-culture experiments. An additional MUC16-knockdown clone (csMUC16neg-OVC-A) was also used in these experiments. This protocol for the generation of this additional cell line has been reported [37]. The three sublines were plated at equal densities in separate wells. After cells were 70% confluent, NKL cells were introduced in the cultures and plates were incubated for 24 h (Figure 8A) or 48 h (Figure 8B). At the end of the incubation, NKL cells and lysed non-adherent ovarian tumor cells were removed and the remaining adherent colonies and individual cells were manually counted. Very few adherent colonies and cells per observed field remained in the csMUC16neg-OVC/NKL and csMUC16neg-OVC-A/NKL co-cultures (Figure 8). However, 3-4-fold more colonies and cells/field were detected in the csMUC16pos-OVC/NKL co-cultures (Figure 8).

Bottom Line: Our results indicate that MUC16-mediated inhibition of immune synapse formation is an effective mechanism employed by ovarian tumors to evade immune recognition.The NK cell leukemia cell line (NKL), which does not express KIRs but are positive for DNAM-1 and NKG2D, also conjugated and lysed MUC16-knockdown cells more efficiently than MUC16 expressing controls.The higher csMUC16 levels on the NKL resistant tumor cells correlated with more protection from lysis as compared to target cells that were never exposed to the effectors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Obstetrics and Gynecology, University of Wisconsin-Madison, Madison, WI, USA.

ABSTRACT

Background: Cancer cells utilize a variety of mechanisms to evade immune detection and attack. Effective immune detection largely relies on the formation of an immune synapse which requires close contact between immune cells and their targets. Here, we show that MUC16, a heavily glycosylated 3-5 million Da mucin expressed on the surface of ovarian tumor cells, inhibits the formation of immune synapses between NK cells and ovarian tumor targets. Our results indicate that MUC16-mediated inhibition of immune synapse formation is an effective mechanism employed by ovarian tumors to evade immune recognition.

Results: Expression of low levels of MUC16 strongly correlated with an increased number of conjugates and activating immune synapses between ovarian tumor cells and primary naïve NK cells. MUC16-knockdown ovarian tumor cells were more susceptible to lysis by primary NK cells than MUC16 expressing controls. This increased lysis was not due to differences in the expression levels of the ligands for the activating receptors DNAM-1 and NKG2D. The NK cell leukemia cell line (NKL), which does not express KIRs but are positive for DNAM-1 and NKG2D, also conjugated and lysed MUC16-knockdown cells more efficiently than MUC16 expressing controls. Tumor cells that survived the NKL challenge expressed higher levels of MUC16 indicating selective lysis of MUC16(low) targets. The higher csMUC16 levels on the NKL resistant tumor cells correlated with more protection from lysis as compared to target cells that were never exposed to the effectors.

Conclusion: MUC16, a carrier of the tumor marker CA125, has previously been shown to facilitate ovarian tumor metastasis and inhibits NK cell mediated lysis of tumor targets. Our data now demonstrates that MUC16 expressing ovarian cancer cells are protected from recognition by NK cells. The immune protection provided by MUC16 may lead to selective survival of ovarian cancer cells that are more efficient in metastasizing within the peritoneal cavity and also at overcoming anti-tumor innate immune responses.

Show MeSH
Related in: MedlinePlus