George Tsao (University of Hong Kong, Hong Kong SAR China)”Establishment of New NPC Xenografts and Cell Lines for EBV Studies” 曹世華
- Int J Cancer Vol 83 1999 Lim (C-6661)
- elaborate on NPC-HeLa hybrid contamination available NPC cell lines (refers to KW Lo’s paper and JV Strong RNA-seq paper)
- Surgical Maxi William
- NPC Xeno43 65M recurrent NPC: The first xenograft line established from clinical tissue (TI，T2，T3，T4，T5，T6，T7 then … no longer passable) (inoculated on the top of mouse kidney)
- NPC Xeno76 stage1，primary NPC (1. EM examination of EBV virus 2 . WGS of Xenograft and cell line )
- For cell line establishment, Tsao suggested the use of 4 μM Y27632, a Rho kinase inhibitor) based on (Liu et al., Am J Pathol, 2012)
- Rapid drop down of EBV EBER copies
- NPC Xeno47, 52F recurrent, take out of FCS，replenish with GF (avoid differentiation)
- C17 spheroids RPMI with 10% FBS + 4 μM Y27632
- NPC38: a EBV negative NPC cell line
- RNAseq
- NRAS Q61R Q61K 2/4 ( think about drugs。)
- TP53 mutation G245D
- KMT2C/KMT2D
- CYLD mutation and BCL3 and Deubiqnitization
- Notes: 徐正龍醫師的NPC Xeno則多為 meta, 他在Oncotarget的paper (Hsu et al., Oncotarget, 2015)
From Ming-Han
- Issue: need to keep these precious samples.
- Method: Xenograft into Nude mouse kidney capsules.
- Problems: (1) loss EBV quickly; (2) stop growing
Previous Results and issues:
- in 20% cases; HPV18 positive!! (Chen et al IJC 1998)
- Before 2008; nearly all NPC cell lines are E7 or E6 HPV positive!!?
- Decide to make new ones in case doing something wrong studies.
Surgery:
- Cut a small hole of nude mice from the back.
- Squeeze the kidney out
- implant 3 small NPC primary tumours
- put back kidney and wait.
Result:
- they found the tumor grows faster and faster if they re-implant the “grow-up” tumor back to new nude mice.
- Successfully rate: (2-3/40 (T2); 2-3/17 (T3))
- For the ones failed to grow up: 1. central parts of tumor are necrosis; 2. highly in lytic replication.
ROCK inhibitor (27632) can make epithelial cells grow indefinitely no matter with tumor or without tumor. (4 uM is the best!!) can grow 1000 days. if using ROCK inhibitor to cultivate NPC cell lines?
- Yes, can grow
- Chromosome become a mass
- 95% EBV+ cell after culture
- P1-P10 (100 EBV copy/cell); >P32 (drop to 10 copy EBV/cell)
- These cells can be injected back to mice
- The removal of Y27632: cell stop growing?? and turn into lytic!!
Further
- PD80 cell line (very old) if plus TPA+SB: still lytic!! can collect virus and infect AkataEBV- cell and induced virus by anti-IgG
- Final have 1 primary cell line NPC47C; around PD90, 100% EBV+.
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Question: try spheroids cell culture? Can they grow into 3D?
Ans: Yes, but grow very slowly.
Finally, sequence the tumor genome before or after Xenografts.
NRAS mutations in NPC? (2/4 in Xenograft, 2/40 in primary NPC)
TP53 mutation higher.
KMT2D, CYLD, TRAF3, P53 higher mutated in Xenograft
Q1: what’s CYLD’s function? Deubiquitin?
A1: Can inhibit NFKB activation and may change the localisation of BCL3 from the nucleus to cytosol. BCL3 also involved in NFKB activation.
Q2: EBV-NPC: only isolated from recurrent NPC; what’s the genomic hallmarks inside?
A2: Huge Huge amount of genomic alteration compared to the fresh-isolated tumours.
Q3: EBV can block ATM pathway
Q4: NPC interestingly can easily recurrent back after 5 years or even longer but H and N tumor usually not. (這點和和信鄭主任所述相同)
Q5: Need to take care whether these Xenograft cell lines be contaminated by murine viruses. (SF: Oh my god!)
EBV Meeting Session 10: Nasopharyngeal carcinoma and other epithelial cell tumors
“Establishment and characterization of newly established nasopharyngeal carcinoma xenografts and in vitro NPC cell lines for EBV studies”
George Tsao, University of Hong Kong, China