Biology subjects

Cunha, A.

Publications and source records attributed to Cunha, A..

4 recordsLinked to original sources

bioRxiv2025

Lactate Blocks Tertiary Lymphoid Structure Formation by Inhibiting B Cell Chemotaxis

Tertiary lymphoid structures (TLS) and B cell infiltration are strong predictors of immunotherapy success across cancers, including triple-negative breast cancer (TNBC). However, immune-cold TNBCs often lack both features. Here, we identify a tumor-intrinsic mechanism that actively suppresses B cell recruitment. Despite evidence of B cell responses in cancer-associated lymph nodes (cLNs), B cells fail to infiltrate TNBC tumors or form TLS. This exclusion is not simply due to chemokine deficiency as exogenous chemokine addition fails to restore B cell migration. Using fractionation and metabolic profiling, we identify lactate as a dominant tumor-secreted metabolite that directly impairs B cell chemotaxis by disrupting mitochondrial metabolism. In vivo, combining lactate inhibition with engineered chemokine secretion promotes cLN-derived B cell infiltration and enables TLS formation, particularly when coupled with CD40 stimulation. Transcriptomics analyses across several human cancer datasets strengthen the association between high glycolytic activity with poor B-cell infiltration in chemokine-rich tumors. Together, our findings reveal lactate as a key metabolic barrier to B cell trafficking and TLS induction, suggesting that metabolic reprogramming may provide an avenue to convert "immune-cold" tumors into TLS-rich, immunologically responsive microenvironments.

cancer biology↗

bioRxiv2024

High-throughput screening of human genetic variants by pooled prime editing

Understanding the effects of rare genetic variants remains challenging, both in coding and non-coding regions. While multiplexed assays of variant effect (MAVEs) have enabled scalable functional assessment of variants, established MAVEs are limited by either exogenous expression of variants or constraints of genome editing. Here, we introduce a pooled prime editing (PE) platform in haploid human cells to scalably assay variants in their endogenous context. We first optimized delivery of variants to HAP1 cells, defining optimal pegRNA designs and establishing a co-selection strategy for improved efficiency. We characterize our platform in the context of negative selection by testing over 7,500 pegRNAs targeting SMARCB1 for editing activity and observing depletion of highly active pegRNAs installing loss-of-function variants. We next assess variants in MLH1 via 6-thioguanine selection, assaying 65.3% of all possible SNVs in a 200-bp region spanning exon 10 and distinguishing LoF variants with high accuracy. Lastly, we assay 362 non-coding MLH1 variants across a 60 kb region in a single experiment, identifying pathogenic variants acting via multiple mechanisms with high specificity. Our analyses detail how filtering for highly active pegRNAs can facilitate both positive and negative selection screens. Accordingly, our platform promises to enable highly scalable functional assessment of human variants.

genomics↗

bioRxiv2019

Identifying relationships between imaging phenotypes and lung cancer-related mutation status: EGFR and KRAS

EGFR and KRAS are the most frequently mutated genes in lung cancer, being active research topics in targeted therapy. Biopsy is the traditional method to genetically characterise a tumour. However, it is a risky procedure, painful for the patient, and, occasionally, the tumour might be inaccessible. This work aims to study and debate the nature of the relationships between imaging phenotypes and lung cancer-related mutation status. Until now, the literature has failed to point to new research directions, mainly consisting of results-oriented works in a field where there is still not enough available data to train clinically viable models. We intend to open a discussion about critical points and to present new possibilities for future radiogenomics studies. We conducted high-dimensional data visualisation and developed classifiers, which allowed us to analyse the results for EGFR and KRAS biological markers according to different combinations of input features. We show that EGFR mutation status might be correlated to CT scans imaging phenotypes, however, the same does not seem to hold true for KRAS mutation status. Also, the experiments suggest that the best way to approach this problem is by combining nodule-related features with features from other lung structures.

bioengineering↗

bioRxiv2018

Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust

In situ hybridization based on the mechanism of hybridization chain reaction (HCR) has addressed multi-decade challenges to imaging mRNA expression in diverse organisms, offering a unique combination of multiplexing, quantitation, sensitivity, resolution, and versatility. Here, with third-generation in situ HCR, we augment these capabilities using probes and amplifiers that combine to provide automatic background suppression throughout the protocol, ensuring that even if reagents bind non-specifically within the sample they will not generate amplified background. Automatic background suppression dramatically enhances performance and robustness, combining the benefits of higher signal-to-background with the convenience of using unoptimized probe sets for new targets and organisms. In situ HCR v3.0 enables multiplexed quantitative mRNA imaging with subcellular resolution in the anatomical context of whole-mount vertebrate embryos, multiplexed quantitative mRNA flow cytometry for high-throughput single-cell expression profiling, and multiplexed quantitative single-molecule mRNA imaging in thick autofluorescent samples.\n\nSUMMARYIn situ hybridization chain reaction (HCR) v3.0 exploits automatic background suppression to enable multiplexed quantitative mRNA imaging and flow cytometry with dramatically enhanced ease-of-use and performance.

developmental biology↗