These findings indicate that fibroblasts redirect chemotherapy responses toward a stress-adaptive, persister-like phenotype and establish fibroblast-containing 3D melanoma spheroids as a physiologically relevant platform for studying tumour microenvironment-mediated chemotherapy tolerance and stromal-tumour interactions.
Abstract
Melanoma is an aggressive malignancy that rapidly adapts to therapy. While chemotherapy resistance has traditionally been attributed to tumour-intrinsic mechanisms, growing evidence implicates the tumour microenvironment in shaping drug tolerance. However, few in vitro models capture the stromal complexity needed to study this interaction. We developed two multicellular melanoma spheroid models of increasing stromal complexity: a baseline model of melanoma, endothelial, and macrophage cells (BEM), and a fibroblast-containing counterpart (BEMF), and compared their transcriptional response to doxorubicin. Fibroblast inclusion increased the doxorubicin concentration required to achieve comparable growth inhibition. While untreated BEMF spheroids exhibited only modest baseline transcriptional differences, they showed a profoundly reshaped transcriptional response after doxorubicin exposure, displaying broader and higher-magnitude changes. These responses were characterized by suppression of proliferative and cell-cycle programmes, together with activation of inflammatory, immune-associated, metabolic, and stress-adaptive pathways. Higher-resolution pathway analyses further revealed coordinated attenuation of mitotic progression, checkpoint regulation, homologous recombination repair, and Rho GTPase signalling, consistent with a shift toward stress-adaptive and phenotypically plastic states, rather than classical resistance mechanisms. Transcriptome-derived transcription factor activity inference supported this regulatory rewiring. Integration with curated resistance-associated genes and external transcriptomic datasets demonstrated strong conservation of core transcriptional features across heterogeneous experimental systems, including consistent suppression of proliferation-associated genes and induction of inflammatory signalling programmes. Together, these findings indicate that fibroblasts redirect chemotherapy responses toward a stress-adaptive, persister-like phenotype and establish fibroblast-containing 3D melanoma spheroids as a physiologically relevant platform for studying tumour microenvironment-mediated chemotherapy tolerance and stromal-tumour interactions.
Abstract Melanoma exhibits dynamic transcriptional adaptations to microenvironmental cues. Phenotypic heterogeneity impacts treatment response, particularly in melanoma brain metastasis (MBM), where therapeutic resistance is common. To interrogate MBM cellular ecosystem, we profiled 114,455 single-cell transcriptomes from publicly available dataset. Melanoma cells were dichotomized by WNT5A expression levels to proliferative and invasive subtypes. Intercellular communication networks were inferred using CellChat, with top predicted interactions validated in vivo and in vitro. Invasive melanoma (WNT5A-high) showed strongest interactions with endothelial cells through laminin-integrin signaling. Proliferative melanoma (WNT5A-low) interacted most strongly with microglia, which induced a shift toward an invasive tumor state through MCAM activation. To assess melanoma phenotype—microenvironment interactions, control and WNT5A-overexpressing cells were injected intracranially into C57BL/6J mice. WNT5A tumors demonstrated significantly faster growth and shorter survival. Histologic examination revealed significantly greater proportion of invasive infiltrates in WNT5A tumors, frequently tracking along native vasculature. In vitro, WNT5A-overexpressing cells showed increased adhesion to endothelial cells and perivascular matrix, and ex vivo brain slice co-cultures demonstrated greater perivascular localization. Immunofluorescent imaging of microglia revealed three morphologies: homeostatic microglia in non‑tumor regions, enlarged intratumoral microglia, and elongated microglia within invasive infiltrates aligned with MCAM‑positive tumor tracks. While immunohistochemistry confirmed higher overall WNT5A expression in WNT5A-overexpressing tumors, melanoma cells within invasive infiltrates of control tumors exhibited comparable WNT5A expression, suggesting local microenvironmental induction of an invasive phenotype. Single-cell RNA sequencing of tumor-associated microglia identified three subclusters, including one with elevated WNT5A expression. Ongoing spatial transcriptomic analyses in human MBM aim to validate the spatial organization of melanoma phenotypes and their interactions with microglia and other brain cell types. Integrated analyses show that MBM exhibits dynamic bidirectional signaling across tumor cells, endothelium, and microglia, collectively driving invasion and proliferation. Targeting these communication networks may offer a promising therapeutic strategy.
Kevin Zhang, Mao Yang, Ming Yuang et al.· Neuro-Oncology Advances· 0 citations
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, CD163high 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.
Göran Landberg, Mikaela Ståhlberg, E. Frisk et al.· Biomedicine & pharmacotherap...· 0 citations
Neuroblastoma (NB) is the most common extracranial solid tumour of childhood and remains a leading cause of paediatric cancer mortality, particularly in high-risk disease driven by MYCN amplification. Although MYCN is a central oncogenic driver, its role as a transcription factor has limited direct therapeutic targeting, shifting attention toward downstream metabolic and microenvironmental dependencies. Increasing evidence indicates that MYCN-driven metabolic rewiring extends beyond tumour-intrinsic processes to reshape the tumour microenvironment (TME), influencing immune composition and stromal dynamics. Recent advances in single-cell and spatial profiling technologies have revealed substantial heterogeneity within the NB TME, highlighting complex interactions between tumour cells, immune populations, and stromal components. Among these, cancer-associated fibroblasts (CAFs) have emerged as key regulators of extracellular matrix architecture, immune modulation, and metabolic crosstalk. However, CAF identity, functional diversity, and lineage relationships in NB remain incompletely defined, with significant overlap between tumour-intrinsic mesenchymal programs and stromal fibroblast signatures. In this review, we synthesise current understanding of MYCN-driven metabolic reprogramming and its impact on CAF heterogeneity and immune regulation. We integrate insights from adult cancers with emerging data in NB to critically evaluate CAF functional states, including inflammatory and myofibroblastic programs, and their roles in shaping tumour progression, immune exclusion, and therapeutic response. By framing NB as a MYCN-remodelled tumour ecosystem, this review identifies key knowledge gaps in stromal biology and highlights the need to resolve CAF heterogeneity and tumour–stroma interactions. These insights have broader implications for MYC-driven malignancies and support the development of integrated therapeutic strategies targeting both tumour cells and their supportive microenvironment.
M. Shiferaw, K. Somers, Z. Kovacevic· Cancer Metastasis Review· 0 citations
Cancer stem cells (CSCs) drive metastasis and therapy resistance, yet their behaviour within the complex tumour microenvironment remains poorly understood. Here we use a fluorescent reporter that marks CSCs to show that CSCs and their more differentiated progeny display strikingly different population dynamics during metastatic lung colonization in breast cancer models. CSC expansion is rapidly curtailed early in colonization, suggesting a strong negative feedback mechanism acting selectively on this subpopulation. We showed that CSCs are exceptionally sensitive to local microenvironmental cues such as cell crowding and nutrient availability. They respond earlier and more extensively than their differentiated progeny, thereby coupling tumour growth to resource and space availability. Microenvironmental signals converge on the transcriptional regulatory complex YAP/TAZ/TEAD, with CSC sensitivity arising from elevated signal reception and greater chromatin accessibility at TEAD-regulated enhancers. Targeting upstream inputs to this pathway reversed chemotherapy-induced CSC enrichment in lung metastases, suggesting a potential therapeutic strategy. Using a dynamic fluorescent reporter, Tang et al. demonstrate that cancer stem cells are hyper-responsive to microenvironmental cues, which they sense and adapt to through YAP/TAZ signalling, thereby playing an essential role in breast cancer lung metastasis.
Binwu Tang, Jacob Minin, V. Gonzalez et al.· Nature Cell Biology· 0 citations
The RAS/mitogen-activated protein kinase (MAPK) pathway is a pivotal oncogenic driver in numerous malignancies, most prominently pancreatic ductal adenocarcinoma, colorectal carcinoma, non-small cell lung cancer, and melanoma. The emergence of direct KRAS inhibitors, pan-RAS inhibitors, and agents targeting downstream signaling pathways (including MEK and ERK) has profoundly reshaped the therapeutic landscape. However, durable clinical responses remain scarce, owing to profound, rapid, and adaptive resistance mechanisms that extend far beyond tumor cell-intrinsic pathways. Over the past five years, accumulating evidence has unequivocally established that the tumor microenvironment (TME)—particularly its immune components—plays a key role in driving this resistance. This review synthesizes recent advances in understanding how specific TME constituents, including immune cells, cancer-associated fibroblasts (CAFs), and the extracellular matrix (ECM), interact with tumor cells to promote escape from pathway inhibition. We examine bidirectional signaling induced by therapy, consequent tumor cell plasticity, and the formation of a protective niche. Finally, we outline rational combination strategies that simultaneously target the RAS/MAPK axis and key TME vulnerabilities, such as immunotherapy combinations, CAF reprogramming, and ECM normalization, to overcome stromal-mediated resistance and achieve deeper, more sustained clinical responses.
Wen-Hao Ma, Xing-Yu Guo, Xiu-Ting Liu· Cancer Advances· 0 citations
Malignant cells, stromal cells, immunological populations, extracellular matrix elements, and other signalling chemicals make up the tumour microenvironment (TME), a dynamic and intricate ecology that controls cancer initiation, development, metastasis, and treatment response. There is growing evidence that the reciprocal interactions between tumour cells and the tumour microenvironment are critical in defining the course of the illness and the effectiveness of therapy. This review aims to recapitulate the molecular, genetic, epigenetic, and immunological determinants governing tumor microenvironment dynamics, emphasising their contributions to therapeutic resistance, the development of microenvironment-targeted therapy approaches, and the progression of cancer. A literature review was conducted using peer-reviewed articles published in high-impact scientific journals. Relevant studies addressing the molecular architecture of the tumor microenvironment, signaling pathways, immune regulation, tumor heterogeneity, therapeutic resistance, and an integrative picture of current knowledge was produced by critically analysing and synthesising developing technology. and future research directions. The evidence demonstrates that growth factor signalling, inflammatory pathways, hypoxia, metabolic reprogramming, genetic and epigenetic alterations, and extracellular vesicle-mediated communication alter the tumour microenvironment, encouraging tumour growth, metastasis, immune escape, and resistance to immunotherapy, chemotherapy, radiation, and targeted treatment. By enhancing biomarker identification and customised therapy decision making, emerging technologies including as single-cell multi-omics, spatial transcriptomics, artificial intelligence, organoid models, and liquid biopsy are boosting precision oncology. Targeting the tumor microenvironment alongside malignant cells offers significant opportunities to overcome therapeutic resistance and improve clinical outcomes. Integrating multi-omics technologies with precision medicine methods are anticipated to aid in the creation of individualised, long-lasting, and more potent cancer treatments.
Dr. Gopalaxmi Nath, Moumita Ghosh, Dr Shahid Ahmad Shergojry et al.· Genetics and Molecular Resea...· 0 citations