Genome-scale functional genomics identify genes preferentially essential for multiple myeloma cells compared to other neoplasias.

Clinical progress in multiple myeloma (MM), an incurable plasma cell (PC) neoplasia, has been driven by therapies that have limited applications beyond MM/PC neoplasias and do not target specific oncogenic mutations in MM. Instead, these agents target pathways critical for PC biology yet largely dispensable for malignant or normal cells of most other lineages. Here we systematically characterized the lineage-preferential molecular dependencies of MM through genome-scale clustered regularly interspaced short palindromic repeats (CRISPR) studies in 19 MM versus hundreds of non-MM lines and identified 116 genes whose disruption more significantly affects MM cell fitness compared with other malignancies. These genes, some known, others not previously linked to MM, encode transcription factors, chromatin modifiers, endoplasmic reticulum components, metabolic regulators or signaling molecules. Most of these genes are not among the top amplified, overexpressed or mutated in MM. Functional genomics approaches thus define new therapeutic targets in MM not readily identifiable by standard genomic, transcriptional or epigenetic profiling analyses.

Genome-scale screens identify factors regulating tumor cell responses to natural killer cells.

To systematically define molecular features in human tumor cells that determine their degree of sensitivity to human allogeneic natural killer (NK) cells, we quantified the NK cell responsiveness of hundreds of molecularly annotated 'DNA-barcoded' solid tumor cell lines in multiplexed format and applied genome-scale CRISPR-based gene-editing screens in several solid tumor cell lines, to functionally interrogate which genes in tumor cells regulate the response to NK cells. In these orthogonal studies, NK cell-sensitive tumor cells tend to exhibit 'mesenchymal-like' transcriptional programs; high transcriptional signature for chromatin remodeling complexes; high levels of B7-H6 (NCR3LG1); and low levels of HLA-E/antigen presentation genes. Importantly, transcriptional signatures of NK cell-sensitive tumor cells correlate with immune checkpoint inhibitor (ICI) resistance in clinical samples. This study provides a comprehensive map of mechanisms regulating tumor cell responses to NK cells, with implications for future biomarker-driven applications of NK cell immunotherapies.

Chemotherapy Induces Senescence-Like Resilient Cells Capable of Initiating AML Recurrence.

Patients with acute myeloid leukemia (AML) frequently relapse after chemotherapy, yet the mechanism by which AML reemerges is not fully understood. Herein, we show that primary AML cells enter a senescence-like phenotype following chemotherapy in vitro and in vivo. This is accompanied by induction of senescence/inflammatory and embryonic diapause transcriptional programs, with downregulation of MYC and leukemia stem cell genes. Single-cell RNA sequencing suggested depletion of leukemia stem cells in vitro and in vivo, and enrichment for subpopulations with distinct senescence-like cells. This senescence effect was transient and conferred superior colony-forming and engraftment potential. Entry into this senescence-like phenotype was dependent on ATR, and persistence of AML cells was severely impaired by ATR inhibitors. Altogether, we propose that AML relapse is facilitated by a senescence-like resilience phenotype that occurs regardless of their stem cell status. Upon recovery, these post-senescence AML cells give rise to relapsed AMLs with increased stem cell potential. SIGNIFICANCE: Despite entering complete remission after chemotherapy, relapse occurs in many patients with AML. Thus, there is an urgent need to understand the relapse mechanism in AML and the development of targeted treatments to improve outcome. Here, we identified a senescence-like resilience phenotype through which AML cells can survive and repopulate leukemia.This article is highlighted in the In This Issue feature, p. 1307.

An Embryonic Diapause-like Adaptation with Suppressed Myc Activity Enables Tumor Treatment Persistence.

Treatment-persistent residual tumors impede curative cancer therapy. To understand this cancer cell state we generated models of treatment persistence that simulate the residual tumors. We observe that treatment-persistent tumor cells in organoids, xenografts, and cancer patients adopt a distinct and reversible transcriptional program resembling that of embryonic diapause, a dormant stage of suspended development triggered by stress and associated with suppressed Myc activity and overall biosynthesis. In cancer cells, depleting Myc or inhibiting Brd4, a Myc transcriptional co-activator, attenuates drug cytotoxicity through a dormant diapause-like adaptation with reduced apoptotic priming. Conversely, inducible Myc upregulation enhances acute chemotherapeutic activity. Maintaining residual cells in dormancy after chemotherapy by inhibiting Myc activity or interfering with the diapause-like adaptation by inhibiting cyclin-dependent kinase 9 represent potential therapeutic strategies against chemotherapy-persistent tumor cells. Our study demonstrates that cancer co-opts a mechanism similar to diapause with adaptive inactivation of Myc to persist during treatment.

Functional Genomics Identify Distinct and Overlapping Genes Mediating Resistance to Different Classes of Heterobifunctional Degraders of Oncoproteins.

Heterobifunctional proteolysis-targeting chimeric compounds leverage the activity of E3 ligases to induce degradation of target oncoproteins and exhibit potent preclinical antitumor activity. To dissect the mechanisms regulating tumor cell sensitivity to different classes of pharmacological "degraders" of oncoproteins, we performed genome-scale CRISPR-Cas9-based gene editing studies. We observed that myeloma cell resistance to degraders of different targets (BET bromodomain proteins, CDK9) and operating through CRBN (degronimids) or VHL is primarily mediated by prevention of, rather than adaptation to, breakdown of the target oncoprotein; and this involves loss of function of the cognate E3 ligase or interactors/regulators of the respective cullin-RING ligase (CRL) complex. The substantial gene-level differences for resistance mechanisms to CRBN- versus VHL-based degraders explains mechanistically the lack of cross-resistance with sequential administration of these two degrader classes. Development of degraders leveraging more diverse E3 ligases/CRLs may facilitate sequential/alternating versus combined uses of these agents toward potentially delaying or preventing resistance.

Pleiotropic Mechanisms Drive Endocrine Resistance in the Three-Dimensional Bone Microenvironment.

Although hormonal therapy (HT) inhibits the growth of hormone receptor-positive (HR+) breast and prostate cancers, HT resistance frequently develops within the complex metastatic microenvironment of the host organ (often the bone), a setting poorly recapitulated in 2D culture systems. To address this limitation, we cultured HR+ breast cancer and prostate cancer spheroids and patient-derived organoids in 3D extracellular matrices (ECM) alone or together with bone marrow stromal cells (BMSC). In 3D monocultures, antiestrogens and antiandrogens induced anoikis by abrogating anchorage-independent growth of HR+ cancer cells but exhibited only modest effects against tumor cells residing in the ECM niche. In contrast, BMSC induced hormone-independent growth of breast cancer and prostate cancer spheroids and restored lumen filling in the presence of HR-targeting agents. Molecular and functional characterization of BMSC-induced hormone independence and HT resistance in anchorage-independent cells revealed distinct context-dependent mechanisms. Cocultures of ZR75-1 and LNCaP with BMSCs exhibited paracrine IL6-induced HT resistance via attenuation of HR protein expression, which was reversed by inhibition of IL6 or JAK signaling. Paracrine IL6/JAK/STAT3-mediated HT resistance was confirmed in patient-derived organoids cocultured with BMSCs. Distinctly, MCF7 and T47D spheroids retained ER protein expression in cocultures but acquired redundant compensatory signals enabling anchorage independence via ERK and PI3K bypass cascades activated in a non-IL6-dependent manner. Collectively, these data characterize the pleiotropic hormone-independent mechanisms underlying acquisition and restoration of anchorage-independent growth in HR+ tumors. Combined analysis of tumor and microenvironmental biomarkers in metastatic biopsies of HT-resistant patients can help refine treatment approaches. SIGNIFICANCE: This study uncovers a previously underappreciated dependency of tumor cells on HR signaling for anchorage-independent growth and highlights how the metastatic microenvironment restores this malignant property of cancer cells during hormone therapy.

Undifferentiated small round cell sarcoma in a young male: a case report.

CIC-rearranged sarcomas (CRSs) have recently been characterized as a distinct sarcoma subgroup with a less favorable prognosis compared to other small round cell sarcomas. CRSs share morphologic features with Ewing's sarcoma and prior to 2013 were grouped under undifferentiated sarcomas with round cell phenotype by the WHO classification. In this report, whole-genome sequencing and RNA sequencing were performed for an adolescent male patient with CRS who was diagnosed with undifferentiated pleomorphic sarcoma (UPS) by three contemporary institutions. Somatic mutation analysis identified mutations in IQGAP1, CCNC, and ATXN1L in pre- and post-treatment tissue samples, as well as a CIC-DUX4 fusion that was confirmed by qPCR and DUX4 immunohistochemistry. Of particular interest was the overexpression of the translation factor eEF1A1, which has oncogenic properties and has recently been identified as a target of the investigational agent plitidepsin. This case may provide a valuable waypoint in the understanding and classification of CRSs and may provide a rationale for targeting eEF1A1 in similar soft tissue sarcoma cases.

The p97 Inhibitor CB-5083 Is a Unique Disrupter of Protein Homeostasis in Models of Multiple Myeloma.

Inhibition of the AAA ATPase, p97, was recently shown to be a novel method for targeting the ubiquitin proteasome system, and CB-5083, a first-in-class inhibitor of p97, has demonstrated broad antitumor activity in a range of both hematologic and solid tumor models. Here, we show that CB-5083 has robust activity against multiple myeloma cell lines and a number of in vivo multiple myeloma models. Treatment with CB-5083 is associated with accumulation of ubiquitinated proteins, induction of the unfolded protein response, and apoptosis. CB-5083 decreases viability in multiple myeloma cell lines and patient-derived multiple myeloma cells, including those with background proteasome inhibitor (PI) resistance. CB-5083 has a unique mechanism of action that combines well with PIs, which is likely owing to the p97-dependent retro-translocation of the transcription factor, Nrf1, which transcribes proteasome subunit genes following exposure to a PI. In vivo studies using clinically relevant multiple myeloma models demonstrate that single-agent CB-5083 inhibits tumor growth and combines well with multiple myeloma standard-of-care agents. Our preclinical data demonstrate the efficacy of CB-5083 in several multiple myeloma disease models and provide the rationale for clinical evaluation as monotherapy and in combination in multiple myeloma. Mol Cancer Ther; 16(11); 2375-86. ©2017 AACR.

NF-κB dysregulation in multiple myeloma.

The nuclear factor-κB (NF-κB) transcription factor family plays critical roles in the pathophysiology of hematologic neoplasias, including multiple myeloma. The current review examines the roles that this transcription factor system plays in multiple myeloma cells and the nonmalignant accessory cells of the local microenvironment; as well as the evidence indicating that a large proportion of myeloma patients harbor genomic lesions which perturb diverse genes regulating the activity of NF-κB. This article also discusses the therapeutic targeting of the NF-κB pathway using proteasome inhibitors, a pharmacological class that has become a cornerstone in the therapeutic management of myeloma; and reviews some of the future challenges and opportunities for NF-κB-related research in myeloma.

Estrogen receptors ß1 and ß2 are associated with distinct responses of estrogen receptor α-positive breast carcinoma to adjuvant endocrine therapy.

Our purpose was to assess whether and how ERß1 and/or ERß2 expression status could predict response of early stage ERα-positive breast carcinoma to adjuvant endocrine therapy (AET). ERß1 and ERß2 expression were determined using immunohistochemistry. ERß1- and ERß2-positivity were derived from receiver operating characteristic analysis and the median percentage of immunostained tumor cells, respectively. Patients with recurrent disease were grouped according to whether they relapsed within 4 years or after 4 years from surgery. The predictive significance of ERß1 and ERß2 was determined using Kaplan-Meier survival analysis and Cox proportional hazards regression analysis. ERß1-positivity in the first-4-year relapse patient group was lower and ERß2-positivity in the post-4-year relapse group was higher compared with no-relapse group. ERß1-positivity was associated with lower tumor size and longer first-4-year disease-free survival, while ERß2-positivity was associated with shorter post-4-year disease-free survival. Cox multivariate analysis including ERß1, ERß2 and established clinico-pathological variables showed that ERß1-positivity was an independent predictor of lower first-4-year risk of relapse. Thus, low ERß1 expression and high ERß2 expression are markers for identification of AET-treated ERα-positive breast carcinoma patients at risk of early and late relapse, respectively.

Prenatal exposure to BPA alters the epigenome of the rat mammary gland and increases the propensity to neoplastic development.

Exposure to environmental estrogens (xenoestrogens) may play a causal role in the increased breast cancer incidence which has been observed in Europe and the US over the last 50 years. The xenoestrogen bisphenol A (BPA) leaches from plastic food/beverage containers and dental materials. Fetal exposure to BPA induces preneoplastic and neoplastic lesions in the adult rat mammary gland. Previous results suggest that BPA acts through the estrogen receptors which are detected exclusively in the mesenchyme during the exposure period by directly altering gene expression, leading to alterations of the reciprocal interactions between mesenchyme and epithelium. This initiates a long sequence of altered morphogenetic events leading to neoplastic transformation. Additionally, BPA induces epigenetic changes in some tissues. To explore this mechanism in the mammary gland, Wistar-Furth rats were exposed subcutaneously via osmotic pumps to vehicle or 250 µg BPA/kg BW/day, a dose that induced ductal carcinomas in situ. Females exposed from gestational day 9 to postnatal day (PND) 1 were sacrificed at PND4, PND21 and at first estrus after PND50. Genomic DNA (gDNA) was isolated from the mammary tissue and immuno-precipitated using anti-5-methylcytosine antibodies. Detection and quantification of gDNA methylation status using the Nimblegen ChIP array revealed 7412 differentially methylated gDNA segments (out of 58207 segments), with the majority of changes occurring at PND21. Transcriptomal analysis revealed that the majority of gene expression differences between BPA- and vehicle-treated animals were observed later (PND50). BPA exposure resulted in higher levels of pro-activation histone H3K4 trimethylation at the transcriptional initiation site of the alpha-lactalbumin gene at PND4, concomitantly enhancing mRNA expression of this gene. These results show that fetal BPA exposure triggers changes in the postnatal and adult mammary gland epigenome and alters gene expression patterns. These events may contribute to the development of pre-neoplastic and neoplastic lesions that manifest during adulthood.

Estrogen receptor ß2 is inversely correlated with Ki-67 in hyperplastic and noninvasive neoplastic breast lesions.

PURPOSE: The purpose of the study is to compare expression levels of ΕRα, ERß1, ERß2 and cell proliferation marker Ki-67 in normal breast and hyperplastic and noninvasive neoplastic breast lesions. MATERIALS AND METHODS: Routinely processed breast tissue from 55 patients provided 65 cases of noninvasive lesions, namely, epithelial hyperplasia of usual type (HUT), apocrine metaplasia (AM), atypical hyperplasia (AH) and ductal carcinoma in situ (DCIS) and 14 cases of adjacent normal breast tissue. Expression of ERα, ERß1 and ERß2 were evaluated using immunohistochemistry and correlated with Ki-67 labeling index (Ki-67 LI) and menopausal status of the patients. RESULTS: Compared with normal breast, ERα expression increased in low to intermediate-grade DCIS (DCIS1/2) and tended to decrease in high-grade DCIS, while ERß1 expression decreased in DCIS irrespective of grade. Mean Ki-67 LI in HUT, low to intermediate-grade DCIS and high-grade DCIS was higher than in normal breast. Higher than normal Ki-67 LI correlated with low ERß2 expression in the whole set of cases and with high ERα expression and low ERß2 expression in the postmenopausal cases of the subset that is generated by excluding AM and high-grade DCIS. Postmenopausal status correlated with low ERß1 expression in the whole set and with higher than normal Ki-67 LI, high ERα expression and low ERß1 expression in the subset. CONCLUSIONS: These findings are in accordance with an ERα-opposing oncosuppressive role of ERß2 in mammary carcinogenesis along the HUT-AH-DCIS1/2 pathway.

Decoding the pathophysiology and the genetics of multiple myeloma to identify new therapeutic targets.

In recent years, significant progress has been achieved in the characterization of the transcriptional profiles, gene mutations and structural chromosomal lesions in myeloma cells. These studies have identified many candidate therapeutic targets, which are recurrently deregulated in myeloma cells. However, these targets do not appear, at least individually, to represent universal driver(s) of this disease. Furthermore, evaluation of these recurrent lesions does not suggest that they converge to a single molecular pathway. Detailed integration of molecular and functional data for these candidate targets and pathways will hopefully dissect which of them play more critical roles for each of the different individual molecular defined subtypes of this disease. This review focuses on how recent updates in our understanding of myeloma pathogenesis and molecular characterization may impact ongoing and future efforts to develop new therapeutics for this disease.

Estrogen receptor beta 2 is associated with poor prognosis in estrogen receptor alpha-negative breast carcinoma.

PURPOSE: Our aim was to examine the prognostic significance of ERbeta1 and ERbeta2 expression in ERalpha-negative breast carcinomas. MATERIALS AND METHODS: We evaluated nuclear and cytoplasmic expression of ERbeta1 and ERbeta2 by immunohistochemistry in a group of 95 patients with long follow-up. ERbeta1 and ERbeta2 status was correlated with clinicopathological parameters and disease outcome. Univariate and multivariate analyses of ERbeta1 and ERbeta2 as independent markers of disease-free survival (DFS) were carried out using the Cox proportional hazards model. RESULTS: Nuclear ERbeta1 (nERbeta1) and nERbeta2 status was positively correlated (p = 0.01). nERbeta1 positivity was associated with low histological grade (p = 0.01) in all patients and in the nERbeta2-positive subgroup (p = 0.03) but not in the nERbeta2-negative (p = 0.27). nERbeta2 positivity was associated with lymph node involvement and tumor relapse in all cases (p < 0.00 and p < 0.00, respectively) and in the nERbeta1-negative subgroup (p < 0.00 and p < 0.00, respectively) but not in the nERbeta1-positive (p = 0.09 and p = 0.20, respectively). nERbeta2 positivity was associated with poor DFS in all patients (log-rank p <0.00), in the post-menopausal patient subgroup (log-rank p = 0.02) and in the HER2-negative (triple-negative) subgroup (log-rank p = 0.04). Cox multivariate analysis including ERbeta1, ERbeta2 and established clinicopathological variables highlighted ERbeta2 as an independent marker of early disease recurrence (hazard ratio 4.87; 95 % confidence interval 1.07-22.3; p = 0.04). CONCLUSION: High nERbeta2 is an independent marker of early relapse in ERalpha-negative breast carcinoma, and in particular, in the nERbeta1-negative, the post-menopausal patient and the triple-negative subgroups. These findings suggest that inhibition of expression and/or function of ERbeta2 could improve disease outcome.

High male chimerism in the female breast shows quantitative links with cancer.

Clinical observations suggest that pregnancy provides protection against cancer. The mechanisms involved, however, remain unclear. Fetal cells are known to enter the mother's circulation during pregnancy and establish microchimerism. We investigated if pregnancy-related embryonic/fetal stem cell integration plays a role in breast cancer. A high-sensitivity Y-chromosome assay was developed to trace male allogeneic cells (from male fetus) in females. Fixed-embedded samples (n = 206) from both normal and breast cancer patients were screened for microchimerism. The results were combined with matching clinicopathological and histological parameters and processed statistically. The results show that in our samples (182 informative) more than half of healthy women (56%) carried male cells in their breast tissue for decades (n = 68), while only one out of five in the cancer sample pool (21%) (n = 114) (odds ratio = 4.75, CI at 95% 2.34-9.69; p = 0.0001). The data support the notion that a biological link may exist between chimerism and tissue-integrity. The correlation, however, is non-linear, since male microchimerism in excess ("hyperchimerism") is also involved in cancer. The data suggest a link between hyperchimerism and HER2-type cancers, while decreased chimerism ("hypochimerism") associates with ER/PR-positive (luminal-type) breast cancers. Chimerism levels that correlate with protection appear to be non-random and share densities with the mammary progenitor components of the stem cell lineage in the breast. The results suggest that protection may involve stem/progenitor level interactions and implicate novel quantitative mechanisms in chimerism biology.

Brentuximab vedotin.

Brentuximab vedotin (SGN-35; Adcetris®) is an anti-CD30 antibody conjugated via a protease-cleavable linker to the potent anti-microtubule agent monomethyl auristatin E (MMAE). Following binding to CD30, brentuximab vedotin is rapidly internalized and transported to lysosomes where MMAE is released and binds to tubulin, leading to cell cycle arrest and apoptosis. Several trials have shown durable antitumor activity with a manageable safety profile in patients with relapsed/refractory Hodgkin lymphoma, systemic anaplastic large cell lymphoma, or primary cutaneous CD30-positive lymphoproliferative disorders. Peripheral sensory neuropathy is a significant adverse event associated with brentuximab vedotin administration. Neuropathy symptoms are cumulative and dose-related. Multiple ongoing trials are currently evaluating brentuximab vedotin alone or in combination with other agents in relapsed/refractory patients, as well as patients with newly diagnosed disease.

Breast epithelial tissue morphology is affected in 3D cultures by species-specific collagen-based extracellular matrix.

Collagen-based gels have been widely used to determine the factors that regulate branching morphogenesis in the mammary gland. The patterns of biomechanical gradients and collagen reorganization influence the shape and orientation of epithelial structures in three-dimensional (3D) conditions. We explored in greater detail whether collagen type I fibers with distinct biomechanical and fiber-assembling properties, isolated from either bovine or rat tail tendon, differentially affected the epithelial phenotype in a tissue culture model of the human breast. Rat tail collagen fibers were densely packed into significantly longer and thicker bundles compared to those of the bovine type (average fascicle length 7.35 and 2.29 µm, respectively; p = 0.0001), indicating increased fiber alignment and biomechanical enablement in the former. MCF10A epithelial cells formed elaborated branched tubular structures in bovine but only nonbranched ducts and acini in rat tail collagen matrices. Ductal branching in bovine collagen was associated with interactions between neighboring structures mediated through packed collagen fibers; these fiber-mediated interactions were absent in rat tail collagen gels. Normal breast fibroblasts increased the final size and number of ducts only in rat tail collagen gels while not affecting branching. Our results suggest that the species of origin of collagen used in organotypic cultures may influence epithelial differentiation into alveolar or ductal structures and the patterns of epithelial branching. These observations underscore the importance of considering the species of origin and fiber alignment properties of collagen when engineering branching organs in 3D matrices and interpreting their role in the tissue phenotype.

The future of antibodies as cancer drugs.

Targeted therapeutics such as monoclonal antibodies (mAbs) have proven successful as cancer drugs. To profile products that could be marketed in the future, we examined the current commercial clinical pipeline of mAb candidates for cancer. Our analysis revealed trends toward development of a variety of noncanonical mAbs, including antibody-drug conjugates (ADCs), bispecific antibodies, engineered antibodies and antibody fragments and/or domains. We found substantial diversity in the antibody sequence source, isotype, carbohydrate residues, targets and mechanisms of action (MOA). Although well-validated targets, such as epidermal growth factor receptor (EGFR) and CD20, continue to provide opportunities for companies, we found notable trends toward targeting less-well-validated antigens and exploration of innovative MOA such as the generation of anticancer immune responses or recruitment of cytotoxic T cells.

World Bispecific Antibody Summit, September 27-28, 2011, Boston, MA.

With more than 30 therapeutic monoclonal antibodies (mAbs) approved and annual global sales of the products at ~$50 billion in 2010, these products have proven to be successful in many ways. Nevertheless, there is room for improvement in performance, and substantial unmet medical needs remain. As a consequence, numerous organizations are devoting resources to engineering novel mAbs such as bispecific antibodies that have increased functionality compared with unmodified IgG molecules. The World Bispecific Antibody Summit, organized by Hanson Wade, drew over 100 participants to Boston to discuss engineering novel bispecific antibodies, generating lead candidates and clinical study and commercialization of the molecules. Approaches such as the trifunctional antibody (TRION), dual variable domain-Ig (Abbott), two-in-one (Genentech), dual affinity retargeting (MacroGenics), kappa-lambda body (NovImmune), bispecific T-cell engager (Micromet) and chemical generation (CovX/Pfizer) were discussed in detail. In addition, posters describing bispecific Affibody® molecules for targeting of EGFR and HER2 (Affibody), T-cell receptor based bi-specifics that target HLA-peptides (Immunocore), a novel mAb-Fv bispecific antibody format utilizing Fc region (Xencore), generation of a tetravalent bispecific antibody against IL4 and IL13 for the treatment of idiopathic pulmonary fibrosis (Sanofi), Combining Affibody® molecules and the Albumod™ technology to create long acting multispecific protein therapeutics (Royal Institute of Technology, Affibody) and COVA301 as a highly potent bispecific inhibitor of IL-17A and TNF-α (Covagen) were presented.

Dual regulation of breast tubulogenesis using extracellular matrix composition and stromal cells.

Epithelial-mesenchymal interactions during embryogenesis are critical in defining the phenotype of tissues and organs. The initial elongation of the mammary bud represents a central morphological event requiring extensive epithelial-mesenchymal crosstalk. The precise mechanism orchestrating this outgrowth is still unknown and mostly animal models have been relied upon to explore this process. Highly tunable three-dimensional (3D) culture models are a complementary approach to address the question of phenotypic determination. Here, we used a 3D in vitro culture to study the roles of stromal cells and extracellular matrix components during mammary tubulogenesis. Fibroblasts, adipocytes, and type I collagen actively participated in this process, whereas reconstituted basement membrane inhibited tubulogenesis by affecting collagen organization. We conclude that biochemical and biomechanical signals mediate the interaction between cells and matrix components and are necessary to induce tubulogenesis in vitro.