The definitive characterization of common cancer stem cell (CSCs) subpopulations in breast cancer subtypes with distinct genotypic and phenotypic features remains an ongoing challenge. In this study, we have used a non-biased genome wide screening approach to identify transcriptional networks that may be specific to the CSC subpopulations in both luminal and basal breast cancer subtypes. In depth studies of three CSC-enriched breast cancer cell lines representing various subtypes of breast cancer revealed a striking hyperactivation of the mevalonate metabolic pathway in comparison to control cells. The upregulation of metabolic networks is a key feature of tumour cells securing growth and proliferative capabilities and dysregulated mevalonate metabolism has been associated with tumour malignancy and cellular transformation in breast cancer. Furthermore, accumulating evidence suggests that Simvastatin therapy, a mevalonate pathway inhibitor, could affect breast cancer progression and reduce breast cancer recurrence. When detailing the mevalonate pathway in breast cancer using a single-cell qPCR, we identified the mevalonate precursor enzyme, HMGCS1, as a specific marker of CSC-enriched subpopulations within both luminal and basal tumour subtypes. Down-regulation of HMGCS1 also decreased the CSC fraction and function in various model systems, suggesting that HMGCS1 is essential for CSC-activities in breast cancer in general. These data was supported by strong associations between HMGCS1 expression and aggressive features, such as high tumour grade, p53 mutations as well as ER-negativity in lymph node positive breast cancer. Importantly, loss of HMGCS1 also had a much more pronounced effect on CSC-activities compared to treatment with standard doses of Simvastatin. Taken together, this study highlights HMGCS1 as a potential gatekeeper for dysregulated mevalonate metabolism important for CSC-features in both luminal and basal breast cancer subtypes. Pharmacological inhibition of HMGCS1 could therefore be a superior novel treatment approach for breast cancer patients via additional CSC blocking functions.

RNA-binding proteins (RBPs) establish the cellular fate of a transcript, but an understanding of these processes has been limited by a lack of identified specific interactions between RNA and protein molecules. Using MS2 RNA tagging, we have purified proteins associated with individual mRNA species induced by osmotic stress, STL1 and GPD1. We found members of the Lsm1-7/Pat1 RBP complex to preferentially bind these mRNAs, relative to the non-stress induced mRNAs, HYP2 and ASH1. To assess the functional importance, we mutated components of the Lsm1-7/Pat1 RBP complex and analyzed the impact on expression of osmostress gene products. We observed a defect in global translation inhibition under osmotic stress in pat1 and lsm1 mutants, which correlated with an abnormally high association of both non-stress and stress-induced mRNAs to translationally active polysomes. Additionally, for stress-induced proteins normally triggered only by moderate or high osmostress, in the mutants the protein levels rose high already at weak hyperosmosis. Analysis of ribosome passage on mRNAs through co-translational decay from the 5’ end (5P-Seq) showed increased ribosome accumulation in lsm1 and pat1 mutants upstream of the start codon. This effect was particularly strong for mRNAs induced under osmostress. Thus, our results indicate that, in addition to its role in degradation, the Lsm1-7/Pat1 complex acts as a selective translational repressor, having stronger effect over the translation initiation of heavily expressed mRNAs. Binding of the Lsm1-7/Pat1p complex to osmostress-induced mRNAs mitigates their translation, suppressing it in conditions of weak or no stress, and avoiding a hyperresponse when triggered. When confronted with external physical or chemical stress, cells respond by increasing the mRNA output of a small number of genes required for stress survival, while shutting down the majority of other genes. Moreover, each mRNA is regulated under stress to either enhance or diminish its translation into proteins. The overall purpose is for the cell to optimize gene expression for survival and recovery during rapidly changing conditions. Much of this regulation is mediated by RNA-binding proteins. We have isolated proteins binding to specific mRNAs induced by stress, to investigate how they affect the stress response. We found members of one protein complex to be bound to stress-induced mRNAs. When mutants lacking these proteins were exposed to stress, ribosomes were more engaged with translating mRNAs than in the wild-type. In the mutants, it was also possible to trigger expression of stress proteins with only minimal stress levels. Tracing the passage of ribosomes over mRNAs, we saw that ribosomes accumulated around the start codon in the mutants. These findings indicate that the protein complex is required to moderate the stress response and prevent it from overreacting, which would be harmful for the cell.

Breast cancer is a heterogeneous disease in terms of cellular and structural composition, and besides acquired aggressive properties in the cancer cell population, the surrounding tumor microenvironment can affect disease progression and clinical behaviours. To specifically decode the clinical relevance of the cancer promoting effects of individual tumor microenvironments, we performed a comprehensive test of 110 breast cancer samples using a recently established in vivo-like 3D cell culture platform based on patient-derived scaffolds (PDSs). Cell-free PDSs were recellularized with three breast cancer cell lines and adaptation to the different patient-based microenvironments was monitored by quantitative PCR. Substantial variability in gene expression between individual PDS cultures from different patients was observed, as well as between different cell lines. Interestingly, specific gene expression changes in the PDS cultures were significantly linked to prognostic features and clinical information from the original cancer. This link was even more pronounced when ERα-status of cell lines and PDSs matched. The results support that PDSs cultures, including a cancer cell line of relevant origin, can monitor the activity of the tumor microenvironment and reveal unique information about the malignancy-inducing properties of the individual cancer niche and serve as a future complementary diagnostic tool for breast cancer.

Recent advances as well as obstacles for immune-based cancer treatment strategies, highlight the notable impact of patient cancer microenvironments on the immune cells and immune targets. Here, we use patient-derived scaffolds (PDS) generated from 110 primary breast cancers to monitor the impact of the cancer microenvironment on immune regulators. Pronounced variation in PD-L1 expression is observed in cancer cells adapted to different patient scaffolds. This variation is further linked to clinical observations and correlated with specific proteins detected in the cell-free PDSs using mass spectrometry. When adding T cells to the PDS-based cancer cultures, the killing efficiency of activated T cells vary between the cultures, whereas non-activated T cells modulate the cancer cell PD-L1 expression to treatment-predictive values, matching killing capacities of activated T cells. Surviving cancer cells show enrichment in cancer stem cell and epithelial-to-mesenchymal transition (EMT) features, suggesting that T cells may not efficiently target cells with metastatic potential. We conclude that clinically relevant insights in how to optimally target and guide immune-based cancer therapies can be obtained by including patient-derived scaffolds and cues from the cancer microenvironment in cancer patient handling and drug development.

Breast cancer progression is intricately influenced by the tumor microenvironment (TME), where cancer-associated fibroblasts (CAFs) play a central role in modulating tumor behavior. To model these interactions, we employed patient-derived scaffolds (PDSs) with preserved native extracellular matrix (ECM) architecture to investigate fibroblast activation and tumor-stroma crosstalk. Mass spectrometry of 63 breast cancer PDSs revealed distinct matrisome clusters associated with tumor grade, recurrence, and CAF-like enrichment, including myofibroblast (myCAFs) and inflammatory CAFs. Normal fibroblasts cultured on PDSs exhibited progressive activation, marked by upregulation of CAF markers (FAP, CD10, PDGFRα), secretory factors (IL6, HGF), and ECM-remodeling genes. Fibroblasts also displayed transcriptional changes linked to cytoskeletal remodeling and cellular plasticity. In transwell co-culture, MDA-MB-231 cells enhanced fibroblast IL6 secretion and altered fibroblast phenotypes, while PDS-grown fibroblasts modulated cancer cell proliferation and stemness transcriptional programs in a subtype-dependent manner. Spatial context also influenced fibroblast activation in direct co-culture. These findings underscore the dynamic, bidirectional nature of tumor-stroma interactions and support PDSs as a physiologically relevant platform for dissecting CAF heterogeneity, ECM-driven modulation, and their potential impact on breast cancer progression.

Breast cancer progression is critically shaped by the tumor microenvironment, yet models that preserve patient-specific microenvironmental complexity remain limited. In particular, how the non-cellular microenvironment regulates macrophage-cancer cell crosstalk and contributes to tumor aggressiveness remains poorly understood. Here, we establish a patient-derived scaffold (PDS) model derived from decellularized breast cancer tissues as a platform to interrogate these interactions. Growth of MDA-MB-231 and MCF-7 cancer cells in PDSs reshaped cytokine gene expression and secretory profiles, leading to transcriptional reprogramming of THP-1 monocytes. Additionally, THP-1 cells were able to infiltrate PDSs and adopted macrophage-like states characterized by increased expression of M2- and pluripotency-related genes, alongside reduced proliferation signatures. Strikingly, transcriptional analysis of THP-1 PDS cultures identified a subset of PDSs derived from estrogen receptor-negative, CD163^high tumors that preferentially induced upregulation of gene programs associated with macrophage differentiation and immunoregulatory gene signatures. Secreted molecules from these THP-1 PDS cultures, in turn, enhanced epithelial-to-mesenchymal transition (EMT)-related and immune-associated gene expression in cancer cells, particularly in MDA-MB-231, revealing transcriptional crosstalk linked to aggressive tumor features. Together, our findings demonstrate that the non-cellular tumor microenvironment preserved in PDSs is sufficient to drive coordinated transcriptional programs in both macrophages and cancer cells. The strong concordance between PDS-induced responses and clinical tumor characteristics underscores the potential of PDS-based models to uncover patient-specific tumor microenvironment interactions and guide future strategies targeting macrophage-cancer cell crosstalk in breast cancer.