Disertasi

Peran lisat trombosit terhadap proliferasi dan marker invasi matrix metalloproteinase-9 dan epithelial-cadherin sel punca kanker payudara CD24 - /CD44 + dalam kaitannya dengan platelet derived growth factor-AB. = The role of platelet lysate on the CD24 - /CD44 + breast cancer stem cell proliferation and cancer invasion marker matrix metalloproteinase-9 and epithelial-cadherin, in the relation with platelet derived growth factor-AB.

Latar Belakang: Trombositosis pada pasien kanker payudara (KPD) diduga berkontribusi pada penyebaran dan sifat invasi sel punca kanker payudara. Modifikasi lingkungan mikro tumor dapat dilakukan untuk meningkatkan efektivitas terapi anti kanker. Belum diketahui apakah platelet derived growth factor (PDGF)-AB dalam lisat trombosit (LT) juga berperan terhadap cancer stem cell (CSC) payudara CD24-/CD44+. Tujuan: Penelitian ini bertujuan untuk menganalisis efek LT dan PDGF-AB didalamnya sebagai lingkungan mikro tumor pada proliferasi dan sifat invasi sel punca kanker payudara CD24-/CD44+ yang ditandai dengan kadar matrix metalloproteinase-9 (MMP-9) dan epithelial-cadherin (E-cadherin). Metode: Penelitian ini merupakan studi eksperimental pada kultur sel punca KPD yang diberi LT dari pasien KPD dan donor sehat. Darah semua donor dilakukan pemeriksaan hematologi dan diproses untuk mendapatkan platelet rich plasma (PRP). Jumlah trombosit per μL PRP setiap donor dihitung. PRP diproses untuk mendapatkan LT. Kadar PDGF-AB LT diukur. LT 0,01% ditambahkan ke dalam medium dulbecco’s modified eagle’s medium (DMEM)-F12 untuk kultur sel punca KPD. Setelah inkubasi 48 jam, total jumlah sel, population doubling time (PDT) dan viabilitas sel dihitung dan dinormalisasikan terhadap nilai kontrolnya. Ekspresi MMP-9 dan E-cadherin dipilih sebagai penanda biologi sifat invasi dan diukur dengan metode enzyme-linked immunosorbent assay (ELISA). Jumlah total sel, PDT, viabilitas sel, kadar MMP-9 dan E-cadherin dibandingkan antara pasien KPD dan donor sehat lalu dianalisis korelasinya dengan jumlah trombosit dan kadar PDGF-AB dalam lisat trombosit. Hasil: Jumlah trombosit dan kadar PDGF-AB dalam LT pasien KPD lebih tinggi dibandingkan LT donor sehat, keduanya dengan nilai p=0,02. LT pasien KPD memicu proliferasi sel punca KPD lebih baik dibandingkan LT donor sehat (p < 0.001) dengan nilai PDT lebih rendah (p=0.001). Proliferasi dan nilai PDT sel punca KPD berkorelasi kuat bermakna dengan kadar PDGF-AB. Hasil tersebut menunjukkan bahwa PDGF-AB berperan dalam proliferasi sel punca KPD. Namun, suplementasi lisat trombosit tidak berefek pada viabilitas sel. Kadar MMP-9 dan E-cadherin paling rendah terukur pada sel punca KPD yang diberi LT pasien KPD. Terdapat korelasi negatif bermakna antara kadar MMP-9 dan E- cadherin dengan kadar PDGF-AB. Kesimpulan: Suplementasi lisat trombosit dari pasien KPD memicu peningkatan proliferasi sel punca KPD serta penurunan E-cadherin, namun belum menunjukkan kenaikan kadar MMP-9.
Kata kunci: E-cadherin, lisat trombosit, matrix metaloproteinase, proliferasi, PRP, sel punca kanker payudara.


Background: Thrombocytosis in breast cancer (BC) patient is supposed to play a role in the invasiveness of breast cancer stem cells. Modification of tumor microenvironment was proposed to increase the efficacy of anticancer therapy. Aim: This study aimed to analyze the effect of platelet lysate (PL) as well as its platelet derived growth factor (PDGF)-AB content as a tumor microenvironment on the CD24-/CD44+ breast cancer stem cell (BCSC) proliferation and invasiveness. Methods: This experimental study treated BCSC culture with PL from BC patients or healthy donors. Venous blood from all donors were subjected to hematology test and processed to obtain PRP. Platelet counts in PRP were determined. PRP was processed to obtain PL. PDGF-AB contents in PL were measured. PL 0.01% was supplemented into dulbecco’s modified eagle’s medium (DMEM)-F12 medium and used for culturing the CD24-/CD44+ BCSCs . After 48 hours, total cell count, population doubling time (PDT), and cell viability were calculated and normalized to its control. Matrix metalloproteinase-9 (MMP-9) and E-cadherin was used as biological marker for CSC invasiveness and measured by enzyme-linked immunosorbent assay (ELISA) method. Total cell count, PDT, cells viability as well as MMP-9 and E-cadherin levels between BCSC, healthy donor platelet lysate and control group were compared and their correlation with platelet count in PRP and PDGF-AB levels in platelet lysates were analyzed. Results: Platelet counts and PDGF-AB levels were higher in BC patient PL compared to healthy donor group, both with a p value of 0.02. BC patient PL could stimulate the proliferation of BCSCs higher than healthy donor PL (p < 0.001) and showed lower PDT value (p=0.001). Cell proliferation and PDT showed strong correlation with PDGF-AB level. This observation suggests that PDGF-AB has a role on BCSC proliferation. PL showed no effect on BCSC viability. MMP-9 and E-cadherin levels were lowest in BCSCs treated with platelet lysate of cancer patient compared to healthy donor and control group. Both MMP-9 and E-cadherin negatively correlated with level of PDGF-AB per mL platelet lysate. Conclusion: Breast cancer patient PL supplementation to BCSC culture medium stimulated BCSC proliferation and suppressed secretion of MMP-9 and E- cadherin in BCSCs in vitro.
Key words: breast cancer stem cells, E-cadherin, matrix metalloproteinase, platelet lysate, proliferation, PRP.