Chemotherapy represents a major treatment modality for cancer, and numerous genetic screens have probed the mechanisms underlying cell-intrinsic resistance or sensitivity to front-line chemotherapy (1
). However, these studies, while informative, have not been adapted to relevant tumor microenvironments, which may contain diverse stromal and/or immune cell types, are subject to immune surveillance, and harbor physical barriers to drug delivery (7
). Additionally, the native tumor microenvironment comprises a diverse mixture of chemokines and cytokines that may impact responses to genotoxic agents (8
). Thus, the central determinants of therapeutic outcome may be highly dependent upon paracrine survival or stress signals. Indeed, it is well documented that gene function and relevance can vary dramatically when compared in vivo
versus in vitro
). Consequently, studying the impact of defined genetic alterations on therapeutic response in native tumor microenvironments is critical for effective drug development, personalized cancer regimens, and the rational design of combination therapies.
Recent advances in the development of tractable mouse models of cancer have, for the first time, enabled the examination of complex sets of defined alterations in individual mice. For example, retroviral infection of murine hematopoietic stem cells or primary embryonic hepatocytes with small pools of short hairpin RNAs (shRNAs), followed by adoptive transfer into lethally-irradiated recipient mice, has been used to screen for suppressors of B cell lymphomagenesis or hepatocellular carcinoma (11
). Additionally, ex vivo
manipulation of lymphoma cells followed by transfer into syngeneic recipient mice has permitted the interrogation of thousands of shRNAs for modulators of tumor growth and dissemination (13
). These screens provide powerful proofs of principle that diverse alterations can be introduced in chimeric tumor models in vivo
and that these systems might permit the simultaneous examination of the relevance of a whole set of genes to therapeutic response in relevant physiological contexts.
Front-line cancer therapies generally exert their effects by modulating the proportion of pro- to anti- apoptotic death regulators, most notably members of the Bcl-2 family (14
). Thus, we reasoned that interrogating Bcl-2 family functionality might provide a high-resolution focus on a crucial facet of cytotoxic cellular responses to chemotherapy in a variety of distinct settings. Notably, previous studies using recombinant BH3 peptides in reconstituted mitochondrial suspensions have systematically identified cellular states associated with the loss of function of one of the BH3-only Bcl-2 family members, the loss of function of a multi-domain pro-apoptotic Bcl-2 family member, or the enhanced function of an anti-apoptotic family member; these states characterize the potential range of dysregulation that the Bcl-2 family can acquire during tumorigenesis and demarcate central cell fate decisions that are susceptible to therapeutic intervention (16
). However, this approach, while quite powerful, does not allow for the comprehensive examination of the role and relevance of individual Bcl-2 family members to cell death following chemotherapy. Here we describe a complementary in vivo
screening approach that provides a detailed assessment of the role of each Bcl-2 family member in the response to chemotherapy in heterogeneous tumor environments.