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Breast Cancer Associated Fibroblast(BCAFs) : Microenviroment. CHUNGNAM NATIONAL UNIVERSITY MEDICAL SCHOOL of Biochemistry PJL Lab. Hong Bum Park Breast cancer and Fibroblast cell line by co-culture on media Tumourmicro-environment elicits innate resistance to RAF inhibitors through HGF secretion Nature Ref#-0001 Clinical and functional significance of loss of caveolin-1 expression in breast cancer-associated fibroblasts J. Pathology Ref#-0002 DMEM High Glucose Sodium Pyruvate GlutaMAX™ Phenol Red MEMa Ribonucleosides Deoxyribonucleosides Phenol Red L-glutamine Minimum Essential Medium (MEM) that contains non-essential amino acids, sodium pyruvate, lipoic acid, vitamin B12, biotin, and ascorbic acid - We show that cancer associated fibroblast co-culture be used DMEM media. -ATCC suggest breast fibroblast cell line was usually EMEM, so we are choice media by DMEM, but primary breast cancer cell was not. - DMEM, MEM and RPMI are different in composition. when we are choose media, we are usually search of on ATCC. If we select wrong media, cells can be change in proliferation, migration ability and morphology. Breast cancer cell lines and Stromal cell lines Tumourmicro-environment elicits innate resistance to RAF inhibitors through HGF secretion Nature Ref#-001 Breast cancer cell line BT-474 EFM192A HCC1419 HCC1569 MCF-7 Breast Breast Breast Breast Breast ATCC DSMZ ATCC ATCC ATCC HTB-20 ACC 258 CRL-2326 CRL-2330 HTB-22 MDA-MB-175-VII MDA-MB-361 SK-BR-3 ZR-75-1 Breast Breast Breast Breast ATCC ATCC ATCC ATCC HTB-25 HTB-27 HTB-30 CRL-1500 Stromal cell line CCD-1065SK skin (breast) ATCC CCD-1068Sk skin (breast) CCD-1069Sk skin (breast) ATCC ATCC HMF Hs343.T PC87322A1 PC87399A1 CRL-2077 CRL-2086 CRL-2089 Breast - Mammary fibroblasts Breast - Mammry CAFs Breast - Mammry CAFs Breast - Mammry CAFs Primary culture, CAFs Primary culture, CAFs Primary culture, CAFs Sciencell ATCC Asterand Asterand 7630 CRL-7245 PC87322A1-CAF PC87399A1-CAF Primary culture CAFs Primary culture, CAFs Primary culture, CAFs 환자의 cancer sample 의 heterogeneity 유지는? Breast cancer cell Marker Scientists have identified a group of surface markers on cells linked to an aggressive type of breast cancer called estrogen receptor-negative canc er. In this preliminary study, estrogen-negative breast cancer developed when three markers, CD44+, CD49fhi, and CD133hi were present sim ultaneously on the surface of human cells taken from breast cancer patients and transplanted into a mouse; this is called a xenograft model. The s cientists named these human cells with tumor-forming ability in mice, xenograft-initiating cells, or XIC. The research, conducted by scientists at the National Cancer Institute (NCI), part of the National Institutes of Health, appeared online May 18, 2010, and in print June 1, 2010, in Cancer Rese arch. National cancer institute http://www.cancer.gov/newscenter/newsfromnci/2010/TripleMarkerENbreastcancer Breast cancer stem cell markers – the rocky road to clinical applications http://breast-cancer-research.com/content/10/5/110 Molecular Profiling of Breast Cancer Cell Lines Defines Relevant Tumor Models and Provide s a Resource for Cancer Gene Discovery 2009 Plus one http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006146?imageURI=info:doi/10.1371/journal.pone.0006146.t001 Significance, detection and markers of disseminated breast cancer cells Endocrine-Related Cancer (2006) 13 1033–1067 http://erc.endocrinology-journals.org/content/13/4/1033.full Breast cancer는 2가지 type로 나눌 수 있다. Luminal, basal type. Luminal type : CD44-/CD24+ Basal type : CD44+/CD24stem/progenitor breast cancer cells can give rise to the heterogeneity of differentiated cells proliferation이 강하게 나타난다. The ability to form mammospheres is a characteristic of cancer stem cells. Tumors are broadly classified histopathologically by expression of either luminal cytokeratins (CK18/19) or basal cytokeratin (CK5), alpha smooth muscle actin (SMA) and vimentin Homogeneous marker : CD29 (beta1-integrin), CD55 (DAF), CD59 (MIRL), CD81 (TAPA1), CD151 (PETA-3), CD166 (ALCAM). CD55 (DAF), and CD59 (MIRL) No expression or low expression : CD66b, CD66c, CD165, and CD324 mesenchymal stem cells : CD44 (H-CAM), CD54 (ICAM), CD90 (Thy-1), CD105 (Endoglin), CD184 (CXCR4), various integrin molecules, such as CD49b (α 2integrin), CD49f (α 6integrin), CD29 (β1integrin), CD61 (β3integrin), and other adhesion molecules including CD54 (ICAM) and CD166 (ALCAM) myoepithelial/basal-like breast cancers (CK5, SMA, and vimentin) luminal tumors (CK18 and CK19) Breast cancer cell Marker Breast cancer cell Marker Significance, detection and markers of disseminated breast cancer cells Endocrine-Related Cancer (2006) 13 1033–1067 http://erc.endocrinology-journals.org/content/13/4/1033.full Breast cancer cell Marker Breast cancer cell Marker Breast cancer cell Marker Breast cancer cell Marker Breast cancer cell Marker Primary Cell Culture into Cancer and Stromal Cell of Tissue Protocol(수정 중) 1. 조직으로부터 4군데 이상 또는 절반 정도 sample을 채취한다.(sample 1g) (sample 대조 : 환자명, 기록 번호) 2. Sample을 RPMI에 넣어 이동을 한다. (RPMI, antibiotic 3. Use HBSS and Sample washing 3 time. 4. 멸균 된 칼로 조직을 분 가량 chopping 한다. 5. collagenase type 2를 시간 처리한다.(360U/ml) 6. c.f.g 를 RPM hr 로 돌려준다. 7. Use HBSS and Sample washing. 8. c.f.g 를 RPM hr 로 돌려준다. 9. RBC 제거를 위해 ficoll을 3:4 비율로 넣어준다. 10. c.f.g를 RPM hr 로 돌려준다. 11. Use HBSS and Sample washing. 12. DMEM을 넣어 37℃ 5% CO2 incubation 시킨다. DMEM Gibco GIB-10569-010 collagenase from colstridium histolyticum type Ⅱ sigma C6885-1G Ficoll-paque plus GE 10014768 Nature Volume: 487, Pages: 500–504 Date published: (26 July 2012) DOI: doi:10.1038 nature11183 Received 07 November 2011 Accepted 04 May 2012 Published online 04 July 2012 Impact Factor : 36,2 NO . 0001 http://www.nature.com/nature/journal/v487/n7408/full/nature11183.html#/suppl ementary-information Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion Ravid Straussman,1 Teppei Morikawa,2 Kevin Shee,1 Michal Barzily-Rokni,1 Zhi Rong Qian,2 Jinyan Du,1 Ashli Davis,1 Margaret M. Mongare,1 Joshua G ould,1 Dennie T. Frederick,3 Zachary A. Cooper,3 Paul B. Chapman,4 David B. Solit,4, 5 Antoni Ribas,6, 7 Roger S. Lo,7, 8 Keith T. Flaherty,3 Shuji Ogino,2, 9 Jennifer A. Wargo3 & Todd R. Golub1, 10, 11, 12 Fibroblast and cancer cell use to Co-culture, they are showed that increase HGF from fibroblast. Inhibition HGF signal (Raf, MET pathway), Melanoma and colorectal cancer line reduce about proliferation. Main actor : PLX4720(Raf inhibitor), PD184352(MEK inhibitor) Many studies have focused on cell-autonomous mechan isms of drug resistance. Oncoprotein-targeted drugs hold enormous promise for the future of cancer treatment.