Extracellular matrix is a key regulator of normal homeostasis and tissue phenotype 1 . Important signals are lost when cells are cultured ex vivo on two-dimensional plastic substrata. Many of these crucial microenvironmental cues may be restored using three-dimensional (3D) cultures of lamininrich extracellular matrix (lrECM) 2 . These 3D culture assays allow phenotypic discrimination between nonmalignant and malignant mammary cells, as the former grown in a 3D context form polarized, growth-arrested acinus-like colonies whereas the latter form disorganized, proliferative and nonpolar colonies3. Signaling pathways that function in parallel in cells cultured on plastic become reciprocally integrated when the cells are exposed to basement membrane-like gels4 -7. Appropriate 3D culture thus provides a more physiologically relevant approach to the analysis of gene function and cell phenotype ex vivo. We describe here a robust and generalized method for the culturing of various human breast cell lines in three dimensions and describe the preparation of cellular extracts from these cultures for molecular analyses. The procedure below describes the 3D 'embedded' assay, in which cells are cultured embedded in an lrECM gel 8 (Fig. 1). By lrECM, we refer to the solubilized extract derived from the Engelbreth-Holm-Swarm mouse sarcoma cells 9 . For a discussion of user options regarding 3D matrices, see Box 1. Alternatively, the 3D 'on-top' assay, in which cells are cultured on top of a thin lrECM gel overlaid with a dilute solution of lrECM, may be used as described in Box 2 ( Fig. 1 and Fig. 2). Materials ReagentsEngelbreth-Holm-Swarm extracellular matrix extract (EHS), growth factor-reduced (Matrigel, BD Biosciences or Cultrex BME, Trevigen) Diaminophenylindole (DAPI)Hard-set mounting medium (VECTASHIELD HardSet, Vector Laboratories or ProLong Gold, Invitrogen) Immunofluorescence (IF) buffer: 0.2% Triton X-100, 0.1% BSA (radioimmunoassay grade), 0.05% Tween 20 in PBS (pH 7.4; sterilized, 0.22 μm filter); for long-term storage, add 7.7 mM NaN 3Correspondence should be addressed to M.J.B. (mjbissell@lbl.gov). Note: Supplementary information is available on the Nature Methods website. Competing Interests Statement:The authors declare no competing financial interests. 3Trypsinize cells from a monolayer to a single-cell suspension.Use cells that are healthy and not more than 75% confluent. 4Aliquot cells to be plated into a 1.5 ml microcentrifuge tube.To perform drug response assays in 3D cultures 7,10,11 ( Fig. 3 and Supplementary Videos 1 and 2 online), compounds may be added to the culture in one of two manners:i. Small-molecule inhibitors: add to medium when cells are plated inStep 6 of the main protocol and Step b of the 3D on-top protocol (Box 2). Include the compound in all media changes for the duration of the culture.ii. Blocking antibodies: mix with cells before plating inStep 5 of the main protocol andStep b of the 3D on-top protocol (Box 2) to ensure complete exposure of the cells to antibodies. Include t...

3D cell cultures are rapidly becoming the method of choice for the physiologically relevant modeling of many aspects of non-malignant and malignant cell behavior ex vivo. Nevertheless, only a limited number of distinct cell types have been evaluated in this assay to date. Here we report the first large scale comparison of the transcriptional profiles and 3D cell culture phenotypes of a substantial panel of human breast cancer cell lines. Each cell line adopts a colony morphology of one of four main classes in 3D culture. These morphologies reflect, at least in part, the underlying gene expression profile and protein expression patterns of the cell lines, and distinct morphologies were also associated with tumor cell invasiveness and with cell lines originating from metastases. We further demonstrate that consistent differences in genes encoding signal transduction proteins emerge when even tumor cells are cultured in 3D microenvironments.

SummaryThe anti-FcRH5/CD3 T cell-dependent bispecific antibody (TDB) targets the B cell lineage marker FcRH5 expressed in multiple myeloma (MM) tumor cells. We demonstrate that TDBs trigger T cell receptor activation by inducing target clustering and exclusion of CD45 phosphatase from the synapse. The dimensions of the target molecule play a key role in the efficiency of the synapse formation. The anti-FcRH5/CD3 TDB kills human plasma cells and patient-derived myeloma cells at picomolar concentrations and results in complete depletion of B cells and bone marrow plasma cells in cynomolgus monkeys. These data demonstrate the potential for the anti-FcRH5/CD3 TDB, alone or in combination with inhibition of PD-1/PD-L1 signaling, in the treatment of MM and other B cell malignancies.

Despite some notable successes cancer remains, for the most part, a seemingly intractable problem. There is, however, a growing appreciation that targeting the tumor epithelium in isolation is not sufficient as there is an intricate mutually sustaining synergy between the tumor epithelial cells and their surrounding stroma. As the details of this dialogue emerge, new therapeutic targets have been proposed. The FDA has already approved drugs targeting microenvironmental components such as VEGF and aromatase and many more agents are in the pipeline. In this article, we describe some of the "druggable" targets and processes within the tumor microenvironment and review the approaches being taken to disrupt these interactions.

Cancer genomes maintain a complex array of somatic alterations required for maintenance and progression of the disease, posing a challenge to identify driver genes among this genetic disorder. Toward this end, we mapped regions of recurrent amplification in a large collection (n ¼ 392) of primary human cancers and selected 620 genes whose expression is elevated in tumors. An RNAi loss-of-function screen targeting these genes across a panel of 32 cancer cell lines identified potential driver genes. Subsequent functional assays identified SHMT2, a key enzyme in the serine/glycine synthesis pathway, as necessary for tumor cell survival but insufficient for transformation. The 26S proteasomal subunit, PSMB4, was identified as the first proteasomal subunit with oncogenic properties promoting cancer cell survival and tumor growth in vivo. Elevated expression of SHMT2 and PSMB4 was found to be associated with poor prognosis in human cancer, supporting the development of molecular therapies targeting these genes or components of their pathways. Cancer Res; 74(11); 3114-26. Ó2014 AACR.