RESUMO
Single-molecule RNA fluorescence in situ hybridization (RNA FISH)-based spatial transcriptomics methods have enabled the accurate quantification of gene expression at single-cell resolution by visualizing transcripts as diffraction-limited spots. While these methods generally scale to large samples, image analysis remains challenging, often requiring manual parameter tuning. We present Piscis, a fully automatic deep learning algorithm for spot detection trained using a novel loss function, the SmoothF1 loss, that approximates the F1 score to directly penalize false positives and false negatives but remains differentiable and hence usable for training by deep learning approaches. Piscis was trained and tested on a diverse dataset composed of 358 manually annotated experimental RNA FISH images representing multiple cell types and 240 additional synthetic images. Piscis outperforms other state-of-the-art spot detection methods, enabling accurate, high-throughput analysis of RNA FISH-derived imaging data without the need for manual parameter tuning.
RESUMO
Cellular responses to environmental stimuli are typically thought to be governed by genetically encoded programs. We demonstrate that melanoma cells can form and maintain cellular memories during the acquisition of therapy resistance that exhibit characteristics of cellular learning and are dependent on the transcription factor AP-1. We show that cells exposed to a low dose of therapy adapt to become resistant to a high dose, demonstrating that resistance was not purely selective. The application of therapy itself results in the encoding of transient gene expression into cellular memory and that this encoding occurs for both transiently induced and probabilistically arising expression. Chromatin accessibility showed concomitant persistence. A two-color AP-1 reporter system showed that these memories are encoded in cis, constituting an example of activating cis epigenetics. Our findings establish the formation and maintenance of cellular memories as a critical aspect of gene regulation during the development of therapy resistance.
RESUMO
Splicing is the stepwise molecular process by which introns are removed from pre-mRNA and exons are joined together to form mature mRNA sequences. The ordering and spatial distribution of these steps remain controversial, with opposing models suggesting splicing occurs either during or after transcription. We used single-molecule RNA FISH, expansion microscopy, and live-cell imaging to reveal the spatiotemporal distribution of nascent transcripts in mammalian cells. At super-resolution levels, we found that pre-mRNA formed clouds around the transcription site. These clouds indicate the existence of a transcription-site-proximal zone through which RNA move more slowly than in the nucleoplasm. Full-length pre-mRNA undergo continuous splicing as they move through this zone following transcription, suggesting a model in which splicing can occur post-transcriptionally but still within the proximity of the transcription site, thus seeming co-transcriptional by most assays. These results may unify conflicting reports of co-transcriptional versus post-transcriptional splicing.
Assuntos
Precursores de RNA , Transcrição Gênica , Animais , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA , Íntrons/genética , Mamíferos/genéticaRESUMO
Despite the promise of immune checkpoint inhibition (ICI), therapeutic responses remain limited. This raises the possibility that standard of care treatments delivered in concert may compromise the tumor response. To address this, we employ tobacco-signature head and neck squamous cell carcinoma murine models in which we map tumor-draining lymphatics and develop models for regional lymphablation with surgery or radiation. We find that lymphablation eliminates the tumor ICI response, worsening overall survival and repolarizing the tumor- and peripheral-immune compartments. Mechanistically, within tumor-draining lymphatics, we observe an upregulation of conventional type I dendritic cells and type I interferon signaling and show that both are necessary for the ICI response and lost with lymphablation. Ultimately, we provide a mechanistic understanding of how standard oncologic therapies targeting regional lymphatics impact the tumor response to immune-oncology therapy in order to define rational, lymphatic-preserving treatment sequences that mobilize systemic antitumor immunity, achieve optimal tumor responses, control regional metastatic disease, and confer durable antitumor immunity.
Assuntos
Neoplasias de Cabeça e Pescoço , Inibidores de Checkpoint Imunológico , Animais , Células Dendríticas , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/genética , Humanos , Imunoterapia , Camundongos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapiaRESUMO
Immune checkpoint blockade (ICB) therapy has revolutionized head and neck squamous cell carcinoma (HNSCC) treatment, but <20% of patients achieve durable responses. Persistent activation of the PI3K/AKT/mTOR signaling circuitry represents a key oncogenic driver in HNSCC; however, the potential immunosuppressive effects of PI3K/AKT/mTOR inhibitors may limit the benefit of their combination with ICB. Here we employ an unbiased kinome-wide siRNA screen to reveal that HER3, is essential for the proliferation of most HNSCC cells that do not harbor PIK3CA mutations. Indeed, we find that persistent tyrosine phosphorylation of HER3 and PI3K recruitment underlies aberrant PI3K/AKT/mTOR signaling in PIK3CA wild type HNSCCs. Remarkably, antibody-mediated HER3 blockade exerts a potent anti-tumor effect by suppressing HER3-PI3K-AKT-mTOR oncogenic signaling and concomitantly reversing the immune suppressive tumor microenvironment. Ultimately, we show that HER3 inhibition and PD-1 blockade may provide a multimodal precision immunotherapeutic approach for PIK3CA wild type HNSCC, aimed at achieving durable cancer remission.