Abstract 4011: Deciphering the clonal evolution of recurrent tumor-initiating cells with a CRISPR/Cas9 genome-wide loss-of-function screen in an orthotopic HNSCC surgical model in vivo
Authors: Nowrouzi Ali , Akbarpour Mahdi , Roh Vincent , Wursthorn Anne , Hiou-Feige Agnés , Knoll Maximillian , Tawk Bouchra , Schwager Christian , Simon Christian , Debus Jürgen , Tolstonog Genrich , Abdollahi Amir 
Published: August 2018

Abstract
Local recurrence and distant metastasis constitutes a major obstacle in the curative treatment of cancer. The clonal evolution and molecular mechanisms governing these processes remain elusive. We utilized the CRISPR/Cas9 based genome-wide GeCKO library stably transduced in head and neck cancer (HNSCC) cells (FaDu) and orthotopically transplanted into submental area of the floor-of-mouth (FOM) of NMRI nude mice to identify candidate genes which drive molecular processes leading to the development of local recurrences. Primary tumor bulks were resected leaving a minimal residual disease (MRD) consisting of few loco-regionally disseminated tumor cells to form loco-regional recurrences and distant metastasis - the recurrent tumor initiating cells (R-TIC). Primary tumors (PT) developed within the first 14 days with well-defined borders. Following surgical resection of the primary tumors recurrences started to develop approximately 21 days post-surgery. The rate of local recurrences was 2-fold higher in mice which were transplanted with the GeCKO library compared to the parental FaDu line. From mice with local recurrence (LR), also the lymph nodes (LN) and the lung metastases (LM) were collected. Following multiplex low cycling quantitative PCR of the guide RNA sequences (gRNA-seq) by NGS, 270 million reads were extracted enabling a comprehensive evaluation of the GeCKO library representation and specific functional gene knockout (KO) in the PT and matching LR, LN and LM. In line with the proposed R-TIC hypothesis, we found a highly clonal evolution of few dominating “trunk” KO cells governing the locally recurrent, LN and distant metastases. In average, we could detect close to 13K gRNA-seq in PT indicating a polyclonal composition of the tumor bulk prior to surgery. In contrast, all recurrent tumors exhibited a highly clonal composition with &gt;95% of the read counts originating from only 2-4 unique gRNA-seq. This indicates that the clonal expansion which has led to the growth of the recurrent tumor is initiated by few clones carrying loss-of-function mutations in 2-4 genes. Interestingly the abundancy of these clones was very low or below NGS detection limit in the corresponding PT and clones which initiated LR were also found in the corresponding LN and LM of the same mice. 14 loss-of function candidate genes were shared in all mice which developed LR post-surgery underlining their importance in mechanistically promoting the initiation of local recurrences. We provide here evidence for the existence and clonal evolution of an R-TIC population in HNSCC. Novel R-TIC specific molecular targets discovered in this study will facilitate rational design of treatment strategies aiming to prevent the development of loco-regional recurrences and distant metastasis.
Citation Format: Ali Nowrouzi, Mahdi Akbarpour, Vincent Roh, Anne Wursthorn, Agnés Hiou-Feige, Maximillian Knoll, Bouchra Tawk, Christian Schwager, Christian Simon, Jürgen Debus, Genrich Tolstonog, Amir Abdollahi. Deciphering the clonal evolution of recurrent tumor-initiating cells with a CRISPR/Cas9 genome-wide loss-of-function screen in an orthotopic HNSCC surgical model in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4011.