Biology subjects

Judson-Torres, R. L.

Publications and source records attributed to Judson-Torres, R. L..

14 recordsLinked to original sources

bioRxiv2026

Evolution of immune infiltration and immune triad formation across early melanoma progression

Tumor-infiltrating lymphocytes (TIL) in melanoma are a clinically important prognostic marker and are routinely used to inform patient care. However, when immune recognition of transformed melanocytes begins and how it evolves during early melanoma progression is not well understood. Using spatial transcriptomics and a unique cohort of patient-matched lesions representing multiple stages of early evolution, we investigate T cells, dendritic cells (DCs) and multi-cell immune "triads" during melanoma initiation. We find that T cell and DC density, triad density, and triad proximity to melanoma cells are elevated in regions of Stage I invasive melanoma as compared to Stage 0 in situ lesions and nevi. Further, we find that triad abundance correlates with both T cell and DC density, but is not predicted by clustering patterns of these cell types over larger spatial scales. We conclude that immune recognition of melanoma and engagement with the lesion significantly increases with progression from Stage 0 to Stage I disease. Further, using nevi that are depth-matched in the dermis to Stage I invasive lesions, we demonstrate this increased immune recognition is not a consequence of dermal invasion alone. This points to a paradigm where immune engagement of invasive melanoma reflects recognition of melanoma cell-intrinsic changes that emerge in Stage I disease.

cancer biology↗

bioRxiv2026

Multi-Modal Kinome Profiling Discovers Mesenchymal-Like Polarity Networks that Underly Directed Hepatocellular Carcinoma Cell Migration

Metastasis and associated therapy resistance remain the principal drivers of cancer related death, and there is a pressing need for a deeper mechanistic understanding and anti-metastatic therapies. For patients that suffer from hepatocellular carcinomas (HCCs), which are the most common primary liver cancers, frequent systemic metastasis results in bleak 5-year survival prognoses of only 4%. To metastasize, carcinoma cells must acquire an invasive phenotype, which typically requires switching from an epithelial-like apical-basal polarity to the front-rear polarity of mesenchymal-like cells. Signaling cues that originate in the tumor microenvironment can activate cellular morphogenic programs that drive polarity switching, like the epithelial-mesenchymal transition (EMT). Protein kinases control most cell signaling pathways and are highly actionable drug targets; however, systematic studies determining the kinases that underly the epithelial-mesenchymal polarity switch (EMPS) are lacking. We developed an assay platform that integrates mass spectrometry (MS)-based kinome profiling, broadly capturing kinase network activity, with chemical genetic screening using selective kinase inhibitors and quantitative phase imaging (QPI), serving as the phenotypic readout. Applying this approach that we dubbed morphokin-MS, to epithelial-like HCC cell lines that we induced to undergo EMPS identified a conserved network of 12 kinases that contributed to HCC cell polarity switching and directed cell migration; MS-based kinome profiling of 17 HCC patient tumors showed that these kinase are frequently upregulated in human tumors. morphokin-MS also revealed that death-associated protein kinase 3 (DAPK3) is one of the principal drivers of the EMPS and directed HCC cell migration. Thus, our mechanistic studies revealed that DAPK3 forms a complex with DAPK1 and filamin-A inter-acting protein 1-like (FILIP1L), which act as scaffold proteins that recruit DAPK3 to the centrosome. Pharmaco-logical and genetic inhibition of the DAPK1-DAPK3-FILIP1L complex blocked centrosome repositioning and microtubule polarization toward the leading edge of mesenchymal-like HCC cells, directed cell migration, and invasion. Our morphokin-MS method and comprehensive kinome profiling data will serve as a valuable resource for the cancer research community; our discovery of an inducible mesenchymal-like DAPK1-DAPK3-FILIP1L polarity complex that controls centrosome positioning in motile HCC cells may lead to the development of novel therapeutics for combatting cancer metastasis.

cancer biology↗

bioRxiv2026

Temporal dynamics improves machine learning-based prediction of cell state from quantitative phase imaging

Cell morphology reflects cell health and can distinguish cell-cycle stage, growth arrest, and distinct pathways of cell death. Live, label-free quantitative phase imaging (QPI) captures these features non-invasively and with high temporal resolution, yet many image-based classifiers rely on single frames and cannot separate states whose differences emerge only over time. How much temporal information is needed, and which architecture best exploits it, remain open questions. We assembled 1,874 QPI timelapse sequences spanning six cell states (interphase, mitosis, cell cycle arrest, apoptosis, ferroptosis, and necroptosis) and compared two-dimensional convolutional neural networks (CNNs) with a three-dimensional (3D) spatiotemporal CNN across increasing frame counts. Accuracy improved as frames were added, with the largest gain between one and three frames. The 2D models saturated beyond three frames, whereas the 3D architecture kept improving, reaching 96.5% accuracy and a 3.5% error rate at eleven frames. The temporal information needed tracked the timescale of each process: mitosis was resolved from a single frame, while ferroptosis benefited most from extended sequences. Overall, these results show that dynamic information, rather than static morphology alone, drives accurate cell-state classification, and that 3D architectures are needed to fully exploit it for label-free dynamic phenotyping.

bioengineering↗

bioRxiv2025

QUILPEN provides independent and label-free single-cell quantification of pigmentation dynamics and organelle content

The relationship between melanogenesis, pigmentation, and melanocyte behavior is complex. In melanocytes, pigmentation is often associated with differentiation, yet mature melanocytes vary in pigment content. In melanoma, pigmentation-linked transcriptional programs may have prognostic value, but visual assessments of tumor pigmentation have yielded inconsistent results. Progress linking pigmentation phenotypes to cell state has been limited by a lack of tools that can directly and dynamically quantify melanin content in live cells. Here we present QUantitative Imaging of Label-free Pigment-associated ENtities (QUILPEN), a label-free multi-modal imaging technique that combines quantitative phase imaging (QPI), quadrant darkfield (QDF), and absorption imaging, to independently capture light that has been transmitted, scattered, and absorbed. This non-destructive method enables live-cell imaging over multiple days without labels. We show absorption as a reliable readout of melanin content, which can be decoupled from melanosome content detected by QDF, which measures scattered light. Applying QUILPEN to melanoma cells before and during repigmentation, we find that melanin content is highly heterogeneous, and that this heterogeneity is reinstated upon repigmentation. Lineage tracking further reveals that melanin synthesis rates are heritable and can be transmitted both symmetrically and asymmetrically. QUILPEN enables real-time quantification of pigmentation dynamics and cell-level heterogeneity. SignificancePigmentation is a defining feature of melanocytic cells and linked to behavior, yet melanins optical properties complicate direct measurement with existing approaches. Taking advantage of the absorbing nature of pigment and scattering properties of organelles, we can independently track pigment and organelle content over time and along cell lineages in a label-free manner to reveal population heterogeneity and patterns of inheritance. Research HighlightsQUILPEN is a label-free, live cell imaging pipeline that independently quantifies pigment and organelles in melanocytic cells. Tracking pigment and organelle content along cell lineages reveals varied inheritance of pigment-associated features. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/683473v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@8d1ef9org.highwire.dtl.DTLVardef@ec6ebdorg.highwire.dtl.DTLVardef@5a7b64org.highwire.dtl.DTLVardef@5b0d80_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Transdermal Delivery of Ultradeformable Cationic Liposomes Complexed with miR211-5p (UCL-211) Stabilizes BRAFV600E+ Melanocytic Nevi

Small non-coding RNAs (e.g. siRNA, miRNA) are involved in a variety of melanocyte-associated skin conditions and act as drivers for alterations in gene expression within melanocytes. These molecular changes can potentially affect the cellular stability of melanocytes and promote their oncogenic transformation. Thus, small RNAs can be considered as therapeutic targets for these conditions, however, their topical delivery to the melanocytes through the epidermal barrier is challenging. We synthesized and extensively evaluated ultradeformable cationic liposome (UCLs) carriers complexed with synthetic microRNAs (miR211-5p; UCL-211) for transdermal delivery to melanocytes. UCL-211 complexes were characterized for their physicochemical properties, encapsulation efficiency, and deformability, which revealed a significant advantage over conventional liposomal carriers. Increased expression of miR211-5p stabilizes melanocytic nevi and keeps them in growth-arrested state. We did a comprehensive assessment of cellular delivery, and biological activity of the miR211-5p carried by UCL-211 in vitro and their permeation through the epidermis of intact skin using ex vivo human skin tissue explants. We also demonstrated, in vivo, that topical delivery of miR211-5p by UCL-211 stabilized BRAFV600E+ nevi melanocytes in a benign nevi state.

pharmacology and toxicology↗

bioRxiv2024

The miR-290 and miR-302 clusters are essential for reprogramming of fibroblasts to induced pluripotent stem cells

The miR-290 and miR-302 clusters of microRNAs are highly expressed in naive and primed pluripotent stem cells, respectively. Ectopic expression of the embryonic stem cell-specific cell cycle regulating (ESCC) family of microRNAs arising from these two clusters dramatically enhances the reprogramming of both mouse and human somatic cells to induced pluripotency. Here, we used genetic knockouts to dissect the requirement for the miR-290 and miR-302 clusters during the reprogramming of mouse fibroblasts into induced pluripotent stem cells (iPSCs) with retrovirally introduced Oct4, Sox2, and Klf4. Knockout of either cluster alone did not negatively impact the efficiency of reprogramming. Resulting cells appeared identical to their embryonic stem cell microRNA cluster knockout counterparts. In contrast, the combined loss of both clusters blocked the formation of iPSCs. While rare double knockout clones could be isolated, they showed a dramatically reduced proliferation rate, a persistent inability to fully silence the exogenously introduced pluripotency factors, and a transcriptome distinct from individual miR-290 or miR-302 mutant ESC and iPSCs. Taken together, our data show that miR-290 and miR-302 are essential yet interchangeable in reprogramming to the induced pluripotent state. Impact StatementThe process by which somatic cell reprogramming yields induced pluripotent stem cells (iPSCs) is incompletely understood. MicroRNAs from the miR-290 and miR-302 clusters have been shown to greatly increase reprogramming efficiency, but their requirement in the process has not been studied. Here, we examine this requirement by genetically removing the miRNA clusters in somatic cells. We discover that somatic cells lacking either, but not both, of these miRNA clusters can form iPSC cells. This work thus provides new important insight into mechanisms underlying reprogramming to pluripotency.

developmental biology↗

bioRxiv2024

Quadrant darkfield (QDF) for label-free imaging of intracellular puncta

SignificanceMeasuring changes in cellular structure and organelles is crucial for understanding disease progression and cellular responses to treatments. A label-free imaging method can aid in advancing biomedical research and therapeutic strategies. AimThis study introduces a computational darkfield imaging approach named quadrant darkfield (QDF) to separate smaller cellular features from large structures, enabling label-free imaging of cell organelles and structures in living cells. ApproachUsing a programmable LED array as illumination source, we vary the direction of illumination to encode additional information about the feature size within cells. This is possible due to the varying level of directional scattering produced by features based on their sizes relative to the wavelength of light used. ResultsQDF successfully resolved small cellular features without interference from larger structures. QDF signal is more consistent during cell shape changes than traditional darkfield. QDF signals correlate with flow cytometry side scatter measurements, effectively differentiating cells by organelle content. ConclusionsQDF imaging enhances the study of subcellular structures in living cells, offering improved quantification of organelle content compared to darkfield without labels. This method can be simultaneously performed with other techniques such as quantitative phase imaging to generate a multidimensional picture of living cells in real-time.

bioengineering↗

bioRxiv2024

CSPG4-targeting CAR-macrophages inhibit melanoma growth

Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of hematological malignancies but has been clinically less effective in solid tumors. Engineering macrophages with CARs has emerged as a promising approach to overcome some of the challenges faced by CAR-T cells due to the macrophages ability to easily infiltrate tumors, phagocytose their targets, and reprogram the immune response. We engineered CAR-macrophages (CAR-Ms) to target chondroitin sulfate proteoglycan 4 (CSPG4), an antigen expressed in melanoma, and several other solid tumors. CSPG4-targeting CAR-Ms exhibited specific phagocytosis of CSPG4-expressing melanoma cells. Combining CSPG4-targeting CAR-Ms with CD47 blocking antibodies synergistically enhanced CAR-M-mediated phagocytosis and effectively inhibited melanoma spheroid growth in 3D. Furthermore, CSPG4-targeting CAR-Ms inhibited melanoma tumor growth in mouse models. These results suggest that CSPG4-targeting CAR-M immunotherapy is a promising solid tumor immunotherapy approach for treating melanoma. STATEMENT OF SIGNIFICANCEWe engineered macrophages with CARs as an alternative approach for solid tumor treatment. CAR-macrophages (CAR-Ms) targeting CSPG4, an antigen expressed in melanoma and other solid tumors, phagocytosed melanoma cells and inhibited melanoma growth in vivo. Thus, CSPG4-targeting CAR-Ms may be a promising strategy to treat patients with CSPG4-expressing tumors.

cancer biology↗

bioRxiv2022

Genetic silencing of AKT induces melanoma cell death

Aberrant activation of the PI3K-AKT pathway is common in melanoma but efforts to drug this pathway have proven largely ineffective in patients. In this study, we observed that pharmacological inhibition of AKT was ineffective whereas genetic silencing of all three AKT paralogs significantly abrogated melanoma cell growth through effects on mTORC signaling. This phenotype could be rescued by overexpression of AKT but was dependent on kinase activity. Interestingly, expression of the serine/threonine kinase SGK1 was increased following genetic suppression of AKT but only expression of activated SGK1 could rescue the lethal effect of AKT knockdown. SGK1 also increases tumor growth and decreases survival in a BRAFV600E-driven mouse model of melanoma. Pharmacological inhibition of SGK and AKT reduced cell proliferation. These results demonstrate that SGK1 can compensate for AKT loss in this context and suggest that dual targeting of SGK1 and AKT may represent a novel therapeutic strategy in this disease. SIGNIFICANCEAlthough the AKT1, 2 & 3 genes encode: Bona fide oncoprotein kinases; well-validated downstream effectors of PI3-lipids and: pleiotropic regulators of numerous aberrant properties of cancer cells, their role in melanoma progression and/or maintenance is poorly understood. Here we explore the effects of genetic or pharmacological inhibitors of AKT1-3 and conclude that combined inhibition of AKT plus the related SGK protein kinases may be required to inhibit melanomagenesis.

cancer biology↗

bioRxiv2020

Phenotypic distribution of the stem-like melanoma initiating state depends on CDKN2A status

Human cancers can exhibit phenotype switching, resulting in cells that are more metastatic or that are more tolerant to treatment. However, the relationship between these aggressive states is not well understood. We investigated the dynamics of phenotypic switching in human melanoma cells by monitoring the fluorescent activity from a transgenic reporter of BRN2 promoter activation. Melanoma cells exhibit heterogeneous BRN2 expression patterns that are reestablished upon isolation and clonal outgrowth. Specifically, stable BRN2 expression was generally inherited over multiple generations while undergoing occasional bidirectional interconversion. We found that BRN2 low cells were required for tumor initiation and metastasis in animal engraftment assays but were more sensitive to targeted BRAF inhibition. In contrast, the BRN2 high state was not tumorigenic, but entry into this state was uniform and persistent among cells tolerant to targeted BRAF therapy. Single-cell RNA sequencing analyses revealed core programs exclusive to either the BRN2 high or low cells, each of which is present in ex vivo tumors, demonstrating the physiological relevance of these states. Our findings emphasize that one challenge of effectively targeting phenotype switching in melanoma as a therapeutic strategy could be balancing distinct aggressive phenotypes so that sensitizing tumors to BRAF inhibition does not inadvertently lead to further dissemination. TeaserUnraveling melanomas shape-shifting behavior: insights into how cancer cells swap between metastasis and drug evasion.

cancer biology↗

bioRxiv2020

Single cell analysis of human site-specific melanocyte differentiation and the decoding of developmental programs in melanoma

In humans, epidermal melanocytes are responsible for skin pigmentation, defense against ultraviolet radiation, and the deadliest common skin cancer, melanoma. While there is substantial overlap in melanocyte development pathways between different model organisms, species dependent differences are frequent and the conservation of these processes in human skin remains unresolved1-3. Thus, the biology of developing and adult human melanocytes remains largely uncharacterized. Here, we used a single-cell enrichment and RNA-sequencing pipeline to study human epidermal melanocytes derived directly from skin, capturing transcriptomes across different anatomic sites, developmental age, sexes, and multiple skin tones. Using donor-matched skin from distinct volar and non-volar anatomic locations, we uncovered subpopulations of melanocytes exhibiting site-specific enrichment that occurs during gestation and persists through adulthood. In addition, we identified human melanocyte differentiation transcriptional programs that are distinct from gene signatures generated from model systems. Finally, we use these programs to define patterns of dedifferentiation that are predictive of melanoma prognosis. Overall, the characterization of human melanocytes fresh from skin revealed new subpopulations, human-specific transcriptional programs, and valuable insights into melanoma dedifferentiation.

cancer biology↗

bioRxiv2020

MicroRNAs Restrain Proliferation in BRAFV600E Melanocytic Nevi

Benign melanocytic nevi frequently emerge when an acquired BRAFV600E mutation triggers unchecked proliferation and subsequent arrest in melanocytes. Recent observations have challenged the role of oncogene-induced senescence in melanocytic nevus formation, necessitating investigations into alternative mechanisms for the establishment and maintenance of proliferation arrest in nevi. We compared the transcriptomes of melanocytes from healthy human skin, nevi, and melanomas arisen from nevi and identified a set of microRNAs as highly expressed nevus-enriched transcripts. Two of these microRNAs - MIR211-5p and MIR328-3p - induced mitotic failure, genome duplication and proliferation arrest in human melanocytes through convergent targeting of AURKB. We demonstrate that BRAFV600E induces a similar proliferation arrest in primary human melanocytes that is both reversible and conditional. Specifically, BRAFV600E expression stimulates either arrest or proliferation depending on the differentiation state of the melanocyte. We report genome duplication in human melanocytic nevi, reciprocal expression of AURKB and microRNAs in nevi and melanomas, and rescue of arrested human nevus cells with AURKB expression. Together, our data describe an alternative molecular mechanism for melanocytic nevus formation that is congruent with both experimental and clinical observations.

cancer biology↗

bioRxiv2020

The genomic landscapes of individual melanocytes from human skin

Every cell in the human body has a unique set of somatic mutations, yet it remains difficult to comprehensively genotype an individual cell. Here, we developed solutions to overcome this obstacle in the context of normal human skin, thus offering the first glimpse into the genomic landscapes of individual melanocytes from human skin. We comprehensively genotyped 133 melanocytes from 19 sites across 6 donors. As expected, sun-shielded melanocytes had fewer mutations than sun-exposed melanocytes. However, within sun-exposed sites, melanocytes on chronically sun-exposed skin (e.g. the face) displayed a lower mutation burden than melanocytes on intermittently sun-exposed skin (e.g. the back). Melanocytes located adjacent to a skin cancer had higher mutation burdens than melanocytes from donors without skin cancer, implying that the mutation burden of normal skin can be harnessed to measure cumulative sun damage and skin cancer risk. Moreover, melanocytes from healthy skin commonly harbor pathogenic mutations, likely explaining the origins of the melanomas that arise in the absence of a pre-existing nevus. Phylogenetic analyses identified groups of related melanocytes, suggesting that melanocytes spread throughout skin as fields of clonally related cells, invisible to the naked eye. Overall, our study offers an unprecedented view into the genomic landscapes of individual melanocytes, revealing key insights into the causes and origins of melanoma.

genomics↗

bioRxiv2018

A machine-learning classifier trained with microRNA ratios to distinguish melanomas from nevi

The use of microRNAs as biomarkers has been proposed for many diseases including the diagnosis of melanoma. Although hundreds of microRNAs have been identified as differentially expressed in melanomas as compared to benign melanocytic lesions, limited consensus has been achieved across studies, constraining the effective use of these potentially useful markers. In this study we quantified microRNAs by next-generation sequencing from melanomas and their adjacent benign precursor nevi. We applied a machine learning-based pipeline to identify a microRNA signature that separated melanomas from nevi and was unaffected by confounding variables, such as patient age and tumor cell content. By employing the ratios of microRNAs that were either enriched or depleted in melanoma compared to nevi as a normalization strategy, the classifier performed similarly across multiple published microRNA datasets, obtained by microarray, small RNA sequencing, or RT-qPCR. Validation on separate cohorts of melanomas and nevi correctly classified lesions with 83% sensitivity and 71-83% specificity, independent of variation in tumor cell content of the sample or patient age.

cancer biology↗