The influence of tamoxifen on normal mouse mammary gland homeostasis.

INTRODUCTION: Lineage tracing using inducible genetic labeling has emerged to be a powerful method for interrogating the developmental fate of cells in intact tissues. A common induction mechanism is the use of tamoxifen-dependent Cre recombinase (CreER and CreERT2), but the effects of tamoxifen at doses normally used in lineage-tracing studies on normal adult mammary gland homeostasis are not known. METHODS: We used flow cytometry and immunostaining of intact glands to determine whether varying doses of tamoxifen skew the distribution and the apoptosis and proliferation status of different types of mammary epithelial cells in vivo. We also examined how tamoxifen influences the number of progenitor and mammary repopulating units (MRUs). RESULTS: Our results indicate that ≥5 mg/25 g body weight of tamoxifen induces a transient increase in cell proliferation and in the number of basal cells in the adult mammary epithelium up to 7 days after tamoxifen administration. However, in the medium term (3 weeks), all doses of tamoxifen≥1 mg/25 g body weight result in a decrease in the number of basal and EpCAM+CD49b- luminal cells and a decrease in progenitor cell function. Tamoxifen at doses≥5 mg/25 g body weight induced a transient increase in caspase-3-mediated apoptotic cell death within the mammary epithelium. However, mammary epithelial cell numbers in all subpopulations were restored to their original levels by 8 weeks. No long-lasting effects of tamoxifen on MRU numbers or on pubertal ductal development were observed. CONCLUSION: Tamoxifen can skew the distribution of mammary cell types in a dose-dependent manner, and thus caution must be taken when interpreting lineage-tracing studies using high doses of tamoxifen, particularly when short-duration analyses of a quantitative nature are being performed.

BEHAV3D: a 3D live imaging platform for comprehensive analysis of engineered T cell behavior and tumor response.

Modeling immuno-oncology by using patient-derived material and immune cell co-cultures can advance our understanding of immune cell tumor targeting in a patient-specific manner, offering leads to improve cellular immunotherapy. However, fully exploiting these living cultures requires analysis of the dynamic cellular features modeled, for which protocols are currently limited. Here, we describe the application of BEHAV3D, a platform that implements multi-color live 3D imaging and computational tools for: (i) analyzing tumor death dynamics at both single-organoid or cell and population levels, (ii) classifying T cell behavior and (iii) producing data-informed 3D images and videos for visual inspection and further insight into obtained results. Together, this enables a refined assessment of how solid and liquid tumors respond to cellular immunotherapy, critically capturing both inter- and intratumoral heterogeneity in treatment response. In addition, BEHAV3D uncovers T cell behavior involved in tumor targeting, offering insight into their mode of action. Our pipeline thereby has strong implications for comparing, prioritizing and improving immunotherapy products by highlighting the behavioral differences between individual tumor donors, distinct T cell therapy concepts or subpopulations. The protocol describes critical wet lab steps, including co-culture preparations and fast 3D imaging with live cell dyes, a segmentation-based image processing tool to track individual organoids, tumor and immune cells and an analytical pipeline for behavioral profiling. This 1-week protocol, accessible to users with basic cell culture, imaging and programming expertise, can easily be adapted to any type of co-culture to visualize and exploit cell behavior, having far-reaching implications for the immuno-oncology field and beyond.

A multispectral 3D live organoid imaging platform to screen probes for fluorescence guided surgery.

Achieving complete tumor resection is challenging and can be improved by real-time fluorescence-guided surgery with molecular-targeted probes. However, pre-clinical identification and validation of probes presents a lengthy process that is traditionally performed in animal models and further hampered by inter- and intra-tumoral heterogeneity in target expression. To screen multiple probes at patient scale, we developed a multispectral real-time 3D imaging platform that implements organoid technology to effectively model patient tumor heterogeneity and, importantly, healthy human tissue binding.

Taxanes trigger cancer cell killing in vivo by inducing non-canonical T cell cytotoxicity.

Although treatment with taxanes does not always lead to clinical benefit, all patients are at risk of their detrimental side effects such as peripheral neuropathy. Understanding the in vivo mode of action of taxanes can help design improved treatment regimens. Here, we demonstrate that in vivo, taxanes directly trigger T cells to selectively kill cancer cells in a non-canonical, T cell receptor-independent manner. Mechanistically, taxanes induce T cells to release cytotoxic extracellular vesicles, which lead to apoptosis specifically in tumor cells while leaving healthy epithelial cells intact. We exploit these findings to develop an effective therapeutic approach, based on transfer of T cells pre-treated with taxanes ex vivo, thereby avoiding toxicity of systemic treatment. Our study reveals a different in vivo mode of action of one of the most commonly used chemotherapies, and opens avenues to harness T cell-dependent anti-tumor effects of taxanes while avoiding systemic toxicity.

Uncovering the mode of action of engineered T cells in patient cancer organoids.

Extending the success of cellular immunotherapies against blood cancers to the realm of solid tumors will require improved in vitro models that reveal therapeutic modes of action at the molecular level. Here we describe a system, called BEHAV3D, developed to study the dynamic interactions of immune cells and patient cancer organoids by means of imaging and transcriptomics. We apply BEHAV3D to live-track >150,000 engineered T cells cultured with patient-derived, solid-tumor organoids, identifying a 'super engager' behavioral cluster comprising T cells with potent serial killing capacity. Among other T cell concepts we also study cancer metabolome-sensing engineered T cells (TEGs) and detect behavior-specific gene signatures that include a group of 27 genes with no previously described T cell function that are expressed by super engager killer TEGs. We further show that type I interferon can prime resistant organoids for TEG-mediated killing. BEHAV3D is a promising tool for the characterization of behavioral-phenotypic heterogeneity of cellular immunotherapies and may support the optimization of personalized solid-tumor-targeting cell therapies.

PTEN Loss in E-Cadherin-Deficient Mouse Mammary Epithelial Cells Rescues Apoptosis and Results in Development of Classical Invasive Lobular Carcinoma.

Invasive lobular carcinoma (ILC) is an aggressive breast cancer subtype with poor response to chemotherapy. Besides loss of E-cadherin, a hallmark of ILC, genetic inactivation of PTEN is frequently observed in patients. Through concomitant Cre-mediated inactivation of E-cadherin and PTEN in mammary epithelium, we generated a mouse model of classical ILC (CLC), the main histological ILC subtype. While loss of E-cadherin induced cell dissemination and apoptosis, additional PTEN inactivation promoted cell survival and rapid formation of invasive mammary tumors that recapitulate the histological and molecular features, estrogen receptor (ER) status, growth kinetics, metastatic behavior, and tumor microenvironment of human CLC. Combined inactivation of E-cadherin and PTEN is sufficient to cause CLC development. These CLCs showed significant tumor regression upon BEZ235-mediated inhibition of PI3K signaling. In summary, this mouse model provides important insights into CLC development and suggests inhibition of phosphatidylinositol 3-kinase (PI3K) signaling as a potential therapeutic strategy for targeting CLC.