Proposal for tumor/met sampling and sequencing

Per conversation with Julien the cost per mouse is ~2K and one mouse yields 3-10 dissectible tumors and a few mets. The GFP system is up and running and in principle we can use it for the purposes of the project.

So for 100K we could get 50 mice and sacrifice them at 4 stages: early, middle, late, very late. Early is when the experience shows that the first lung tumors appear. Middle - around the time the first mets appear. Late - multiple mets. Very late - close to death.

This way we would get ~12 mice per time point and a fairly large number of tumors (50*~5=250 tumors and 200-400 mets).

We can then sequence deeply the late ones and do targeted resequencing of the intermediate stages focusing on all of the mutations that we found in the late stages. We can also do RNAseq on them.

Julien, any thoughts?

Julien's Description of the Mouse Model

Lung cancer is the number one cause of cancer deaths in the world. Lung cancer is divided into two major histopathological groups: non-small cell lung cancer (NSCLC, ~80-85% of cases) and small cell lung cancer (SCLC~15-20% of cases). SCLC is the deadliest subtype of lung cancer, with a 5-year survival of only 5% (this number has not changed since the 1970s). SCLC is characterized by the rapid expansion and metastasis of small cells with neuroendocrine features (Fischer and Arcaro, 2008; Rudin et al., 2008).

SCLC is challenging to study in patients because it is often detected late and because of the inherent complex genetic and environmental diversity of these patients. Furthermore, SCLC patients rarely undergo surgery (because surgery does not improve their survival) and primary human material is very scarce. At the genomic level, carcinogens in cigarette smoke induce a number of mutations, making it difficult to distinguish driving mutations (similar to melanoma and UV-induced mutations) (Lee et al., 2010).

To circumvent these limitations, we have developed pre-clinical mouse models of human SCLC, by deleting the Rb and p53 tumor suppressor genes via adenoviral delivery of the Cre recombinase specifically in the lung epithelium (intratracheal injection) of adult conditional mutant mice. This model is based on the observation that tumor cells in more than 90% of human SCLCs are mutant for both the p53 and RB tumor suppressor genes. More recently, we have developed a novel SCLC mouse model bearing Rb, p53 and p130 (an Rb family member) deletions. This triple-knockout mouse develops SCLC more rapidly (and more metastases), providing a powerful system to investigate the molecular and cellular basis of SCLC development (Schaffer et al., 2010).

We have used these mouse models to identify the cell of origin of the disease (Park et al., 2011a), to identify serum biomarkers for early detection (Taguchi et al., 2011), and to investigate the role of the Hedgehog pathway in disease development (Park et al., 2011b).

In the course of these experiments, we have performed a number of microarrays on mouse tumors and, in collaboration with the group of Roman Thomas (Cologne University), we have begun to look at exome sequencing data and CGH arrays (unpublished data).

Given the deadly nature of this cancer type, its highly metastatic behavior, the difficulty to work with human material, and the absence of treatment, we believe that our mouse models of SCLC provide an exciting system to investigate how tumors evolve. Thus, we propose to perform high-throughput sequencing of tumors and metastases and analyze the genome of cells in these cancerous lesions.

Note that sequencing full tumors in the lung might reveal association of tumor cells with specific microbes, as was recently shown in colon cancer.

Note also that we have ways to visualize tumors and mets, using a luciferase reporter, which allows us to monitor tumor development in real-time.

Note finally that we are developing a system where a GFP reporter is turned on only in tumor cells, which may allow to sort these cells from the complex micro-environment in which they grow, as well as from the blood.

Fischer, B., and Arcaro, A. (2008). Current status of clinical trials for small cell lung cancer. Rev Recent Clin Trials 3, 40-61.

Lee, W., Jiang, Z., Liu, J., Haverty, P.M., Guan, Y., Stinson, J., Yue, P., Zhang, Y., Pant, K.P., Bhatt, D., et al. (2010). The mutation spectrum revealed by paired genome sequences from a lung cancer patient. Nature 465, 473-477.

Park, K.S., Liang, M.C., Raiser, D.M., Zamponi, R., Roach, R.R., Curtis, S.J., Walton, Z., Schaffer, B.E., Roake, C.M., Zmoos, A.F., et al. (2011a). Characterization of the cell of origin for small cell lung cancer. Cell Cycle 10.

Park, K.S., Martelotto, L.G., Peifer, M., Sos, M.L., Karnezis, A.N., Mahjoub, M.R., Bernard, K., Conklin, J.F., Szczepny, A., Yuan, J., et al. (2011b). A crucial requirement for Hedgehog signaling in small cell lung cancer. Nat Med.

Rudin, C.M., Hann, C.L., Peacock, C.D., and Watkins, D.N. (2008). Novel systemic therapies for small cell lung cancer. J Natl Compr Canc Netw 6, 315-322.

Schaffer, B.E., Park, K.S., Yiu, G., Conklin, J.F., Lin, C., Burkhart, D.L., Karnezis, A.N., Sweet-Cordero, E.A., and Sage, J. (2010). Loss of p130 accelerates tumor development in a mouse model for human small-cell lung carcinoma. Cancer Res 70, 3877-3883.

Taguchi, A., Politi, K., Pitteri, S., Lockwood, W., Faça, V., Kelly-Spratt, K., Wong, C., Zhang, Q., Chin, A., Park, K., et al. (2011). Lung cancer signatures in plasma based on proeome profiling of mouse tumor models. Cancer Cell Accepted for publication.