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Supplementary Figure Legends. Legends for Supplementary Figures S1-S6.

Supplementary Figures S1-S6. Representative dot plots of in vitro inhibition of T cell activation by TPIN-SCs (S1); Non-irradiated TPIN-SCs arrest in vitro T cell activation (S2); TPIN-SCs inhibit restimulation of antigen-experienced T cells but do not affect fully activated T cells (S3); TPIN-SC-conditioned T cells are hyporesponsive (S4); Silencing of TNC modulates the inhibitory activity of TPIN-SCs (S5); CSCs migration is mediated by the CXCR4/CXCL12 axis (S6).

Supplementary Methods and References. Description of additional methods and procedures used in the study. Also includes Supplementary References.

Supplementary Table S2. Kegg enrichment of proteins upregulated in T cells cultured in the presence of TPIN-SCs.

Supplementary Table S1. Proteins identified and quantified in the SILAC experiment, comparing T cells inhibited by TPIN-SCs (CD62Lhi) with those activated by TNE-SCs (CD62Llo) or with the subset activated by TPIN-SCs (CD62Llo).

Precociously disseminated cancer cells may seed quiescent sites of future metastasis if they can protect themselves from immune surveillance. However, there is little knowledge about how such sites might be achieved. Here, we present evidence that prostate cancer stem–like cells (CSC) can be found in histopathologically negative prostate draining lymph nodes (PDLN) in mice harboring oncogene-driven prostate intraepithelial neoplasia (mPIN). PDLN-derived CSCs were phenotypically and functionally identical to CSC obtained from mPIN lesions, but distinct from CSCs obtained from frank prostate tumors. CSC derived from either PDLN or mPIN used the extracellular matrix protein Tenascin-C (TNC) to inhibit T-cell receptor–dependent T-cell activation, proliferation, and cytokine production. Mechanistically, TNC interacted with α5β1 integrin on the cell surface of T cells, inhibiting reorganization of the actin-based cytoskeleton therein required for proper T-cell activation. CSC from both PDLN and mPIN lesions also expressed CXCR4 and migrated in response to its ligand CXCL12, which was overexpressed in PDLN upon mPIN development. CXCR4 was critical for the development of PDLN-derived CSC, as in vivo administration of CXCR4 inhibitors prevented establishment in PDLN of an immunosuppressive microenvironment. Taken together, our work establishes a pivotal role for TNC in tuning the local immune response to establish equilibrium between disseminated nodal CSC and the immune system. Cancer Res; 75(10); 2095–108. ©2015 AACR.

Large-scale sequencing studies are rapidly identifying putative oncogenic mutations in human tumors. However, discrimination between passenger and driver events in tumorigenesis remains challenging and requires in vivo validation studies in reliable animal models of human cancer. For these reasons, new technologies are needed to expand the genetic toolbox of cancer biologists and allow a more rapid and systematic in vivo interrogation of gene perturbations. In this regard, the advent of CRISPR/Cas9 technologies for somatic genome editing has already paved the way for a new generation of non-germline animal tumor models. For example, liver-specific gene disruption was achieved by transient delivery of components of the CRISPR/Cas9 system in the tail veins of mice, leading to hepatocellular carcinoma (Xue et al., 2014; Weber et al., 2015). Similar approaches have been used to deliver targeted oncogenic mutations to the lung (Platt et al., 2014; Sánchez-Rivera et al., 2014), brain (Zuckermann et al., 2015), and pancreas (Chiou et al., 2015). We have previously shown that the mammary tissue is also amenable to somatic gene editing using intraductal injection of lentiviral vectors encoding Cre recombinase, the CRISPR/Cas9 system, or both in the mammary glands of female mice carrying conditional predisposing alleles (Annunziato et al., 2016). This approach was successfully applied to validate in vivo candidate tumor suppressors implicated in invasive lobular carcinoma (Kas et al., 2017), but it is conceivable that more breast cancer subtypes can be modeled via somatic engineering in mice with distinct predisposing mutations. In this study, we show how somatic engineering via intraductal injection of lentiviruses can be used to develop innovative mouse models of triple-negative breast cancer (TNBC), one of the subtypes with the poorest prognosis in the clinics. Using intraductal injection of Cre-encoding lentiviruses in mice with conditional Brca1 and Trp53 alleles (B1P), we were able to target TNBC-initiating cells from the basal compartment, and induce tumors that retained all the hallmark features of the transgenic Cre-driven tumor counterparts, including latency, histopathology, and DNA copy-number variation (CNV) pattern. Moreover, we could dramatically accelerate tumorigenesis when lentiviral Cre was injected in B1PM mice that harbored, in addition to conditional Brca1 and Trp53 alleles, a conditional knock-in allele overexpressing the Myc oncogene, a candidate driver in BRCA1-associated cancers. Alternatively, somatic Myc expression and loss of BRCA1 and p53 was induced in the mammary glands of B1P mice via intraductal injection of lentiviral vectors encoding Myc and Cre under a viral promoter. In both cases the resulting tumors displayed a reshaped CNV profile that strongly implicated a limited number of recurrent focal amplifications and deletions in the tumorigenic cascade. We reiterated the process by lentiviral overexpression of these candidate driver oncogenes and Cre in the mammary glands of B1PM mice, or by achieving in situ CRISPR/Cas9-mediated somatic gene disruption of one or multiple candidate tumor-suppressor genes in Cas9-transgenic WB1P-Cas9 mice. This versatile somatic platform allows for rapid, flexible, and multiplexable in vivo testing of putative oncogenic hits and deals with the need for increasingly complex mouse models while avoiding the bottlenecks of classical transgenesis. Notably, we show here that we can extend the applicability of non-germline breast cancer modeling in mice to produce genetically and histologically accurate TNBC, which allows in vivo investigation of the underlying biology as well as rapid generation of tailored preclinical models for this aggressive breast cancer subtype. Citation Format: Stefano Annunziato, Julian R. de Ruiter, Chiara S. Brambillasca, Sjors M. Kas, Federica Ferrante, Catrin Lutz, Bjorn Siteur, Bas van Gerwen, Marieke van de Ven, Martine H. van Miltenburg, Linda Henneman, Jos Jonkers. Somatic engineering of the mammary gland for the development of novel mouse models of triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A01.

Breast cancer is the most common cancer among women in the Western world. Approximately 8% of all breast cancer cases may be caused by inheritance of mutations in breast cancer susceptibility genes. Of these, BRCA1 is the most important one, contributing to approximately half of all familial breast cancer cases. BRCA1-related breast tumors show a triple-negative (TBNC) basal like phenotype, that correlates with aggressive characteristics and bad prognosis. A number of BRCA1 associated genes have been identified; among those MYC overexpression seems to have an important role in aggressive TBNC. In line with this, we found MYC to be frequently amplified in both mouse and human BRCA1-deficient breast cancers. Thus, evaluating the contribution of MYC to BRCA1-associated breast cancer might unravel new mechanistic insights and potentially open the way to new therapeutic approaches. To pursue this, we generated a WAPCre;Brca1F/F;Trp53F/F;Myc (WB1P-MYC) mouse model to monitor tumor development. Whereas the WAPCre;Brca1F/F;Trp53F/F (WB1P) mouse model develops tumors after 300 days, Cre-conditional overexpression of the MYC transgene leads to a dramatic acceleration in tumor development with a median survival of 100 days. Tumors of both models were histopathologically classified as solid carcinomas. Further characterization revealed that WB1P-MYC tumors show higher levels of apoptosis and DNA damage. Moreover, we also observed a strong difference in immune infiltrate between WB1P and WB1P-MYC tumors. To better characterize these differences between WB1P and WB1P-MYC tumors we obtained tumor organoids. Importantly, these tumor organoids maintained characteristics of the original tumor like Brca1, P53 deletion status and MYC overexpression. Moreover, they can be orthotopically transplanted in vivo, leading to formation of solid carcinomas. Therefore, they represent a versatile platform to test new drug combinations both in vitro and in vivo. To test if MYC overexpression is also required for maintenance of established tumors, we are developing a “double-layered” system for Cre-conditional and doxycycline-regulatable overexpression of the MYC gene in the WAPCre;Brca1F/F;Trp53F/F model. By administering doxycycline we will induce MYC only in Cre-expressing mammary epithelial cells. After tumor formation MYC will be switched-off to assess its role in tumor maintenance and progression. Furthermore, we would like to test new therapeutic approaches, like combining Myc down-regulation with administration of PARP inhibitors. In addition, tumor organoids of this inducible WB1P-MYC model will be a powerful system to study the MYC-associated phenotype, drugs combinations and forward genetic screens. Citation Format: Chiara Svetlana Brambillasca, Linda Henneman, Julian de Ruiter, Anne Paulien Drenth, Jos Jonkers. Dissecting the role of MYC in BRCA1-associated breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 889.

Purpose. Objectives of this study were to investigate if prostate cancer stem/initiating cells (CSC) obtained from autochthonous tumors possess the molecular characteristics that allow their in vitro and in vivo recognition by cells of the innate and/or adaptive arms of the immune system, and if CSC are source of antigens for the induction of tumor-specific immune responses. Material and Methods. CSC lines established from the prostate of transgenic adenocarcinoma of the mouse prostate (TRAMP) mice were assessed in vitro for the expression of tumor-associated antigens and susceptibility to NK and T cell killing. We also investigated NK and T cells immune surveillance against CSC by measuring the frequency of CSC-induced tumors in immunocompetent and selectively immunodeficient mice. Finally, we assessed if vaccination with dendritic cells pulsed with CSC induced a tumor-specific immune response able to delay the growth of both transplantable and autochthonous prostate tumors. Results. CSC expressed prostate cancer associated antigens, MHC I and MHC II molecules and ligands for natural killer (NK) cells. Indeed, CSC were targets of NK and cytotoxic T lymphocytes both in vitro and in vivo. Vaccination with dendritic cells pulsed with apoptotic CSC induced a tumor-specific immune response that delayed tumor growth in mice challenged with prostate CSC, and caused tumor regression in TRAMP mice. Conclusions. CSC are targets of both innate and adaptive immune responses and could be exploited for the design of novel immunotherapeutic approaches against cancer. Citation Format: Elena Jachetti, Stefania Mazzoleni, Matteo Grioni, Alessia Ricupito, Chiara Brambillasca, Luca Generoso, Arianna Calcinotto, Massimo Freschi, Anna Mondino, Rossella Galli, Matteo Bellone. Prostate cancer stem/initiating cells are targets of both innate and adaptive immunity and elicit potent immune responses against autochthonous prostate tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2613. doi:10.1158/1538-7445.AM2013-2613