The complex interplay between metal abundance, transport mechanisms, cell distribution, and tumor progression-related biological pathways (e.g. metabolism, collagen remodeling) remains poorly understood. Traditionally, genes and metals have been studied in isolation, limiting insights into their interactions. Recent advances in spatial transcriptomics and elemental profiling now enable comprehensive exploration of tissue-wide metal-gene interactions, though integration remains challenging. In this proof-of-concept study, we investigated metal-dependent signaling within the tumor microenvironment of a unique colorectal cancer (CRC) tumor. We implemented a spatial multimodal workflow which integrated elemental imaging, gene expression, cellular composition, and histopathological features to uncover metals-related pathways through spatially resolved gene expression correlation analyses. Preliminary findings revealed significant associations, for instance: elevated iron correlated with mesenchymal phenotypes located at the tumor's proliferative front, correlating with expression of genes involved in the epithelial-to-mesenchymal transition pathways, and extracellular matrix remodeling. Preliminary observations from this single sample revealed that high copper concentrations were localized to regions of active tumor growth and were associated with increased expression of immune response genes. This proof-of-concept workflow demonstrates the feasibility of integrating elemental imaging with spatial transcriptomics to identify metals-based gene correlates. Future application of this workflow to larger patient cohorts will pave the way for expansive comparisons across the metallome and transcriptome, ultimately identifying novel targets for tumor progression biomarkers and therapeutic interventions.

Characterization of PDX model CTG-3346

Despite adjuvant treatment with endocrine therapies, estrogen receptor-positive (ER+) breast cancers recur in a significant proportion of patients. Recurrences are attributable to clinically undetectable endocrine-tolerant persister cancer cells that retain tumor-forming potential. Therefore, strategies targeting such persister cells may prevent recurrent disease. Using CRISPR-Cas9 genome-wide knockout screening in ER+ breast cancer cells, we identified a survival mechanism involving metabolic reprogramming with reliance upon mitochondrial respiration in endocrine-tolerant persister cells. Quantitative proteomic profiling showed reduced levels of glycolytic proteins in persisters. Metabolic tracing of glucose revealed an energy-depleted state in persisters, in which oxidative phosphorylation was required to generate ATP. A phase II clinical trial was conducted to evaluate changes in mitochondrial markers in primary ER+/HER2- breast tumors induced by neoadjuvant endocrine therapy (NCT04568616). In an analysis of tumor specimens from 32 patients, tumors exhibiting residual cell proliferation after aromatase inhibitor-induced estrogen deprivation with letrozole showed increased mitochondrial content. Genetic profiling and barcode lineage tracing showed that endocrine-tolerant persistence occurred stochastically without genetic predisposition. Pharmacologic inhibition of mitochondrial complex I suppressed the tumor-forming potential of persisters in mice and synergized with the antiestrogen drug fulvestrant to induce regression of patient-derived xenografts. These findings indicate that mitochondrial metabolism is essential in endocrine-tolerant persister ER+ breast cancer cells and warrant the development of treatment strategies to leverage this vulnerability for treating breast cancer.Significance: Persister cancer cells that survive endocrine therapy exhibit increased energetic dependence upon mitochondria for survival and tumor regrowth potential, indicating that therapies targeting this metabolic dependency could help prevent disease recurrence.

Supplementary Figure S15- Individual growth curves of WHIM16 tumors from mice treated with a second cycle of E2 ± olaparib.

Generating balanced populations of CD8+ effector and memory T cells is necessary for immediate and durable immunity to infections and cancer. Yet, a definitive understanding of how a diverse CD8+ T cell repertoire differentiates remains unclear. We identified several hundred T cell receptor (TCR) clonotypes that constitute the polyclonal response against a single antigen and found that a majority of TCR clonotypes were highly biased toward memory or effector fates. TCR-intrinsic biases were not stochastic and were dominant over environmental cues. Differential gene expression analysis of memory- or effector-biased TCR clonotypes showed bifurcation of differential fates at the early effector stage. Additionally, phylogenetic analysis revealed that memory-biased clonotypes retain their fate preferences in subclonal populations but effector-biased subclones can switch to a memory fate. Our study highlights that the polyclonal CD8+ T cell response is a composite of unbiased and biased clonotypes with varying capacity to incorporate environmental cues in their cell fate decisions.

Supplementary Figure S13- E2 and olaparib combine to increase R-loop formation.

Supplementary Figure S4- ER overexpression converts E2 from a growth promoter to a growth suppressor.

Supplementary Table S2. Results of single-sample gene set enrichment analysis (ssGSEA) of transcriptomic profiles from HCC-1428 and HCC-1428/LTED cells.

Table S1. Results of genome-wide CRISPR/Cas9 screening of HCC-1428/LTED cells.

Systemic sclerosis (SSc) remains a challenging and enigmatic systemic autoimmune disease, owing to its complex pathogenesis, clinical and molecular heterogeneity, and the lack of effective disease-modifying treatments. Despite a century of research in SSc, the interconnections among microvascular dysfunction, autoimmune phenomena and tissue fibrosis in SSc remain unclear. The absence of validated biomarkers and reliable animal models complicates diagnosis and treatment, contributing to high morbidity and mortality. Advances in the past 5 years, such as single-cell RNA sequencing, next-generation sequencing, spatial biology, transcriptomics, genomics, proteomics, metabolomics, microbiome profiling and artificial intelligence, offer new avenues for identifying the early pathogenetic events that, once treated, could change the clinical history of SSc. Collaborative global efforts to integrate these approaches are crucial to developing a comprehensive, mechanistic understanding and enabling personalized therapies. Challenges include disease classification, clinical heterogeneity and the establishment of robust biomarkers for disease activity and progression. Innovative clinical trial designs and patient-centred approaches are essential for developing effective treatments. Emerging therapies, including cell-based and fibroblast-targeting treatments, show promise. Global cooperation, standardized protocols and interdisciplinary research are vital for advancing SSc research and improving patient outcomes. The integration of advanced research techniques holds the potential for important breakthroughs in the diagnosis, treatment and care of individuals with SSc. The diagnosis, treatment and management of systemic sclerosis (SSc) remains challenging, owing to the complexity of this disease. In this Perspective, an international group of experts discuss the future of SSc research and how the advent of innovative technologies will advance research into and understanding of SSc.

Supplementary Figure S8- Individual tumor volumes from mice treated ± E2.

Supplementary Figure S11- E2 stimulation induces DNA replication stress.

Supplementary Figure S5- ER-negative BT-20 cells do not exhibit increased DNA damage upon E2 treatment, while MCF-7/LTED show weak response.

Supplementary Figure S10- Representative IHC images from WHIM16 and CTG-3346 tumors.

Supplementary Figure S12- ER overexpression promotes E2-induced R-loop formation.

Supplementary Figure S14- Individual growth curves of tumors from mice treated ± E2 and/or olaparib.

Supplementary Fig. 1 CONSORT flow diagram of study patients. Among the 23 patients consented for study participation, 19 were considered evaluable.

Supplementary Figure S1- E2 induces changes in overlapping sets of genes in HCC-1428 and HCC-1428/LTED cells.