Emilia Peuhu

Cell–cell communication in tissues is influenced by contact geometry and molecular signalling. Here, we demonstrate that epithelial intercellular filopodia can penetrate neighbouring cells to form micrometre-long, double-membrane “impact sites” and, in some instances, initiate trans-endocytosis. Using super-resolution live imaging and three-dimensional electron microscopy in cell models, xenografts, and patient-derived tissues, we observe widespread intercellular filopodia and extensive interdigitation at the cell-cell interface. Filopodia impact sites in the recipient cell recruit PACSIN2 into dynamic “PACSIN2 fingers” and are specifically enriched for caveolin-1 and dynamin-2, while clathrin and CLIC pathway markers are not enriched. Most contacts are transient and resolved by retraction, but some undergo scission, with recipient cells internalising filopodial tips across both homotypic and heterotypic interactions, including cancer–endothelium contacts. Correlative light and FIB–SEM analysis reveals that internalised tips are often double-membraned, closely associated with endoplasmic reticulum tubules, and can traffic to lysosomes. These findings define a protrusion-driven, curvature-dependent uptake pathway at cell–cell interfaces and identify filopodia as exchange organelles in epithelial collectives.

Breast cancer is hallmarked by phenotypic transitions enabling abnormal cell proliferation and invasion. The stress-protective transcription factor heat shock factor 2 (HSF2) is associated with cancer, but its function in breast carcinogenesis remains poorly understood. Analysis of human breast tumor samples and mouse in vivo xenografts uncovered that HSF2 expression and activity undergo dynamic changes as a function of tumor progression. HSF2 expression, nuclear localization, and coexpression with the proliferation marker Ki67 are increased in ductal carcinoma in situ (DCIS), suggesting that HSF2 designates hyperplastic cells underlying tumor expansion. In mouse xenografts, HSF2 localization switches from nuclear to cytoplasmic upon DCIS-to-invasive transition. Using cell-based models, we identify canonical transforming growth factor–β (TGF-β) signaling as the molecular mechanism regulating HSF2. TGF-β–mediated down-regulation of HSF2 allowed acquisition of an invasive cell phenotype, which was counteracted by ectopic HSF2. Together, we propose that HSF2 acts as a stage-specific switch between proliferation and invasion in breast cancer.

The KRAS oncogene drives many common and highly fatal malignancies. These include pancreatic, lung, and colorectal cancer, where various activating KRAS mutations have made the development of KRAS inhibitors difficult. Here we identify the scaffold protein SH3 and multiple ankyrin repeat domain 3 (SHANK3) as a RAS interactor that binds active KRAS, including mutant forms, competes with RAF and limits oncogenic KRAS downstream signalling, maintaining mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) activity at an optimal level. SHANK3 depletion breaches this threshold, triggering MAPK/ERK signalling hyperactivation and MAPK/ERK-dependent cell death in KRAS-mutant cancers. Targeting this vulnerability through RNA interference or nanobody-mediated disruption of the SHANK3–KRAS interaction constrains tumour growth in vivo in female mice. Thus, inhibition of SHANK3–KRAS interaction represents an alternative strategy for selective killing of KRAS-mutant cancer cells through excessive signalling.

The three-dimensional (3D) structure of the ductal epithelium and the surrounding extracellular matrix (ECM) are integral aspects of the breast tissue, and they have important roles during mammary gland development, function and malignancy. However, the architecture of the branched mammary epithelial network is poorly recapitulated in the current in vitro models. 3D bioprinting is an emerging approach to improve tissue-mimicry in cell culture. Here, we developed and optimized a protocol for 3D bioprinting of normal and cancerous mammary epithelial cells into a branched Y-shape to study the role of cell positioning in the regulation of cell proliferation and invasion. Non-cancerous cells formed continuous 3D cell networks with several organotypic features, whereas the ductal carcinoma in situ (DCIS) –like cancer cells exhibited aberrant basal polarization and defective formation of the basement membrane (BM). Quantitative analysis over time demonstrated that both normal and cancerous cells proliferate more at the branch tips compared to the trunk region of the 3D-bioprinted cultures, and particularly at the tip further away from the branch point. The location-specific rate of proliferation was independent of TGFβ signaling but invasion of the DCIS-like breast cancer cells was reduced upon the inhibition of TGFβ. Thus, our data demonstrate that the 3D-bioprinted cells can sense their position in the branched network of cells and proliferate at the tips, thus recapitulating this feature of mammary epithelial branching morphogenesis. In all, our results demonstrate the capacity of the developed 3D bioprinting method for quantitative analysis of the relationships between tissue structure and cell behavior in breast morphogenesis and cancer.

The progression of noninvasive ductal carcinoma in situ to invasive ductal carcinoma for patients with breast cancer results in a significantly poorer prognosis and is the precursor to metastatic disease. In this work, we have identified insulin-like growth factor–binding protein 2 (IGFBP2) as a potent adipocrine factor secreted by healthy breast adipocytes that acts as a barrier against invasive progression. In line with this role, adipocytes differentiated from patient-derived stromal cells were found to secrete IGFBP2, which significantly inhibited breast cancer invasion. This occurred through binding and sequestration of cancer-derived IGF-II. Moreover, depletion of IGF-II in invading cancer cells using small interfering RNAs or an IGF-II–neutralizing antibody ablated breast cancer invasion, highlighting the importance of IGF-II autocrine signaling for breast cancer invasive progression. Given the abundance of adipocytes in the healthy breast, this work exposes the important role they play in suppressing cancer progression and may help expound upon the link between increased mammary density and poorer prognosis.

: The major lactiferous ducts of the human breast branch out and ultimately end at terminal ductal lobular units (TDLUs). These glandular structures are the source of milk upon lactation, and also the most common origin of breast cancer. Despite their functional and clinical importance, the three dimensional (3D) architecture of TDLUs has remained undetermined, largely due to their absence in rodent animal models. By utilizing recent technological advances in optical tissue clearing, 3D reconstruction histology and microscopy, we imaged the 3D structure of healthy human breast tissue to determine whether general branching patterns or cell type specific niches exist in the TDLU. Our data demonstrate that highly branched TDLUs, also exhibiting elevated proliferation, are uncommon in the breast tissue regardless of donor age, parity or hormonal contraception. Overall, TDLUs have a consistent shape and their branch parameters are largely comparable between different TDLUs and individuals irrespective of donor age or parity. Simulation of TDLU branching morphogenesis in 3D by mathematical modelling suggests that evolutionarily conserved mechanisms regulate mammary gland branching in humans and mice, despite their anatomical differences. The data also demonstrate a new level of organization within the TDLU structure identifying a main subtree that dominates in bifurcation events and exhibits a more duct-like keratin expression pattern. In all, our data provide the first structural insights into 3D human breast anatomy and branching, and exemplify the power of volumetric imaging in deeper understanding of human breast biology. Graphical

Ductal carcinoma in situ (DCIS) is a pre-invasive stage of breast cancer, where the tumor is encapsulated by a basement membrane (BM). At the invasive phase, the BM barrier is compromised enabling tumor cells to escape into the surrounding stroma. The molecular mechanisms that establish and maintain an epithelial BM barrier in vivo are poorly understood. Myosin-X (MYO10) is a filopodia-inducing motor protein implicated in metastasis and poor clinical outcome in patients with invasive breast cancer (IBC). We compared MYO10 expression in patient-matched normal breast tissue and DCIS lesions and found elevated MYO10 expression in DCIS samples, suggesting that MYO10 might facilitate the transition from DCIS to IBC. Indeed, MYO10 promoted the formation of filopodia and cell invasion in vitro and positively regulated the dissemination of individual cancer cells from IBC lesions in vivo. However, MYO10-depleted DCIS xenografts were, unexpectedly, more invasive. In these xenografts, MYO10 depletion compromised BM formation around the lesions resulting in poorly defined tumor borders and increased cancer cell dispersal into the surrounding stroma. Moreover, MYO10-depleted tumors showed increased EMT-marker-positive cells, specifically at the tumor periphery. We also observed cancer spheroids undergoing rotational motion and recruiting BM components in a filopodia-dependent manner to generate a near-continuous extracellular matrix boundary. Taken together, our data identify a protective role for MYO10 in early-stage breast cancer, where MYO10-dependent tumor cell protrusions support BM assembly at the tumor-stroma interface to limit cancer progression, and a pro-invasive role that facilitates cancer cell dissemination at later stages. Abstract Figure: Highlights: - Filopodia sculpt the tumor-proximal stroma in pre-invasive ductal carcinoma in situ (DCIS). - Filopodia-dependent basement membrane (BM) assembly limits invasive transition of DCIS-like tumors in vivo . - Loss of MYO10-dependent filopodia impairs BM assembly and induces an EMT-like phenotype at the tumor-stroma interface in vivo . - MYO10 filopodia are anti-invasive in DCIS but facilitate dissemination in invasive breast cancer.