Rosano, D. | Search bioRxiv

bioRxiv2026

X-CODE: a dual RNA barcoding system for multi-platform clonal tracking and spatial phenotyping

Experimental dissection of clonal dynamics in complex tissues requires barcoding systems that are scalable, compatible with different analytical platforms, providing phenotypic and spatial resolution. Here we introduce X-CODE, a dual-expressed RNA barcoding system designed to enable high-complexity clonal tracking across sequencing-based, cytometric, and spatial imaging modalities within a unified experimental framework. X-CODE combines a combinatorial, probe-detectable long RNA barcode with a matched short sequencing barcode, enabling seamless integration of probe-based readouts with sequencing and barcode-guided clonal retrieval. We demonstrate robust X-CODE detection by mass cytometry and imaging-based platforms, including spatial RNA barcode readout using via a repurposed Akoya PhenoCycler-Fusion protocol. In addition, we show compatibility with MALDI mass spectrometry imaging for co-registration of clonal and metabolic information. We further demonstrate the feasibility of X-CODE detection within probe-based spatial transcriptomics using the 10x Genomics Xenium platform. Applied to an in vivo model of androgen deprivation in prostate cancer, X-CODE reveals clonal architecture, selection and clone-specific phenotypic and metabolic plasticity underlying castration resistance. Together, X-CODE provides a flexible and broadly accessible platform for integrated clonal analysis across spatial, phenotypic, and molecular dimensions.

cancer biology↗

bioRxiv2021

Unperturbed dormancy recording reveals stochastic awakening strategies in endocrine treated breast cancer cells

Hormone dependent breast cancer (HDBC) is the most commonly diagnosed tumor type in women. Adjuvant endocrine therapies (ET) have been the cornerstone in the clinical management of HDBC patients for over forty years. A vast proportion of HDBC patients incur long periods of clinical dormancy following ET, with tumour awakening appearing at a steady pace for up to 25 years (Pan et al., 2017). Extensive genomic studies have demonstrated that 15-30% of clinical relapses develop recurrent genomic changes which contribute to drug resistance (i.e. ESR1 activating mutations) (Bertucci et al., 2019; Magnani et al., 2017; Razavi et al., 2018). However, even in these cases, there is no conclusive evidence around the pre-existence vs. de novo nature of these events. We previously showed that ETs can trigger and select for dormancy in subpopulations of breast cancer(Hong et al., 2019). In this work we took two novel approaches to investigate the dormancy and awakening roadmap of HDBC cells in unprecedented detail. Firstly, we leveraged a rare cohort of n=5 patients which were treated with primary adjuvant ETs in the absence of surgery (TRACING-HT) to dissect the contribution of genomic aberrations to tumor awakening. Next, we developed a first of its kind evolutionary study in vitro to systematically annotate cancer cells adaptive strategies at single cell level in unperturbed systems during a period of several months (TRADITIOM). Collectively our data suggest that ETs steer HDBC cells into an inherently unstable dormant state. Over time, routes to awakening emerge sporadically and spontaneously in single lineages. Each dormant cell retains an intrinsic awakening probability which we propose is a function of epigenetic decay. Awakening occurs without an external trigger and involves multiple apparent endpoint phenotypes that cannot be fully explained by conventional Darwinian genetic selection processes. Finally, our data show that common genetic hits associated with resistance happen downstream of awakening. Overall, our data have uncovered previously unsuspected roles for stochastic nongenetic events during dormancy with profound clinical implications.

cancer biology↗