Biology subjects

Martinez-Paniagua, M.

Publications and source records attributed to Martinez-Paniagua, M..

4 recordsLinked to original sources

bioRxiv2022

Metabolic plasticity of T-cell therapies: multi-omic profiling of interacting human tumor-infiltrating lymphocytes and autologous tumor adoptive cell therapy

Adoptive cell therapy (ACT) based on ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable antitumor responses even in heavily pretreated patients. However, only a subset of patients responds to ACT; efforts to identify correlates of response have focused on profiling the tumor or the TIL but rarely in an interactive environment. Interactive profiling can provide unique insights into the clinical performance of TILs since the fate, function, and metabolism of TILs are influenced by autologous tumor-derived factors. Here, we performed a suite of cell-sparing assays dubbed holistic analysis of the bioactivity of interacting T cells and autologous tumor cells (HABITAT). HABITAT profiling of TILs used for human ACT and their autologous tumor cells included function-based single-cell profiling by timelapse imaging microscopy in nanowell grids (TIMING); multi-omics using RNA-sequencing and proteomics; metabolite inference using genome-scale metabolic modeling, and pulse-chase assays based on confocal microscopy to profile the uptake and fate of fatty acids (FA). Phenotypically, the ACT TILs from both responders (Rs) and nonresponders (NRs) were comprised of predominantly effector memory T cells (TEM cells) and did not express a high frequency of programmed death ligand-1 (PD-L1) and showed no differences in TCR diversity. Our results demonstrate that while tumor cells from both Rs and NRs are efficient at uptaking FAs, R TILs are significantly more efficient at utilizing FA through fatty acid oxidation (FAO) than NR TILs under nutrient starvation conditions. While it is likely that lipid and FA uptake is an inherent adaptation of TIL populations to lipid-rich environments, performing FAO sustains the survival of TILs and allows them to sustain antitumor cytolytic activity. We propose that metabolic plasticity enabling FAO is a desirable attribute of human TILs for ACT leading to clinical responses.

immunology↗

bioRxiv2022

Integrated single-cell functional and molecular profiling of extracellular vesicle secretion in metastatic breast cancer

Extracellular vesicles (EVs) regulate the tumor microenvironment by facilitating transport of biomolecular cargo including RNA, protein, and metabolites. The biological effects of EV-mediated transport have been studied using supra-physiological concentrations of EVs, but the cells that are responsible for EV secretion and the mechanisms that support EV secretion are not well characterized. We developed an integrated method based on arrays of nanowells to identify individual cells with differences in EV secretion and used an automated robot to perform linked single-cell RNA-sequencing on cloned single cells from the metastatic breast cancer cell line, MDAMB231. Gene expression profiles of clonal cells with differences in EV secretion were analyzed, and a four-gene signature of breast cancer EV secretion was identified: HSP90AA1, HSPH1, EIF5, and DIAPH3. We functionally validated this gene signature by testing it across different cell lines with different metastatic potential demonstrating that the signature correlated with levels of EV secretion. Analysis of the TCGA and METABRIC datasets showed that this signature is associated with poor survival, more invasive breast cancer types, and reduced CD8+ T cell infiltration in human tumors. We anticipate that our method for directly identifying the molecular determinants of EV secretion will have broad applications across cell types and diseases.

cancer biology↗

bioRxiv2022

T-cell potential for CD19-expressing malignancies revealed by multi-dimensional single-cell profiling

Adoptive immunotherapy with T cells expressing chimeric antigen receptors (CARs) for B-cell malignancies serves as a model for identifying subsets with superior clinical activity. We profiled the infusion products (IP) of 16 patients with large B-cell lymphoma (LBCL) using an integrated suite of single-cell assays to reveal the therapeutic potential of CD19-specific CAR+ T cells. Timelapse imaging microscopy in nanowell grids (TIMING) profiling revealed that T cells from responders showed migration (persistent motion for at least one body length), and migration was associated with serial killing capacity. In addition, confocal microscopy revealed that migration is linearly correlated with both mitochondrial volume and lysosomal volume; and scRNA-seq demonstrated that T cells from responders were enriched in pathways related to T-cell killing, migration and actin cytoskeleton, and TCR clustering. A marker-free sorting strategy enriched T cells with migratory capacity and validated serial killing, bioenergetics, and in vivo efficacy. In aggregate, we demonstrate that migration is a cell-intrinsic biomarker independent of CAR design or biomanufacturing, desired in the bioactivity of CAR+ T cells associated with clinical antitumor efficacy.

immunology↗

bioRxiv2020

Functional single-cell profiling identifies that exosomes are associated with increased immune cell infiltration in non-metastatic breast cancer

Exosomes mediate intercellular communication in health and disease. Conventional assays are limited in profiling exosomes secreted from large populations of cells and are unsuitable for studying the functional consequences of individual cells exhibiting varying propensity for exosome secretion. In cancer, since exosomes can support the development of the pre-metastatic niche, cells with varying abilities to secrete exosomes can directly impact tumorigenesis. Here, we developed a high throughput single-cell technique that enabled the mapping of exosome secretion dynamics. By utilizing clinically relevant models of breast cancer, we established that non-metastatic cancer cells secrete more exosomes than metastatic cancer cells. Single-cell RNA-sequencing confirmed that pathways related to exosome secretion were enriched in the non-metastatic cells compared to the metastatic cells. We established isogenic clonal cell lines from non-metastatic cells with differing propensities for exosome secretion and showed that exosome secretion is an inheritable property preserved during cell division. Combined in vitro and in vivo studies with these cell lines suggested that exosome secretion can impede tumor formation. In human non-metastatic breast tumors, tumors with higher secretion of exosomes have a better prognosis, higher immune cytolytic activity, and enrichment of pro-inflammatory macrophages compared to tumors with lower secretion of exosomes. Our single-cell methodology can become an essential tool that enables the direct integration of exosome secretion with multiple cellular functions.

cancer biology↗