Biology subjects

Montalvo, M. J.

Publications and source records attributed to Montalvo, M. J..

4 recordsLinked to original sources

bioRxiv2023

Video Diffusion models for the apoptosis forcasting

Reliable and early prediction of cell death (apoptosis) is critically important in various areas of biology, particularly in characterizing the effectiveness of cell-based infusion products utilized for cancer immunotherapy. While deep Convolutional Neural Networks (CNNs) are often used in state-of-the-art approaches for apoptosis classification, they typically focus solely on individual cells and ignore cell-cell interaction. To address this limitation, we propose a novel generative approach based on a video diffusion model, which predicts future cellular behaviors for early detection of apoptosis events, even before molecular markers like Annexin-V or visual indications like membrane blebbing become apparent. Our approach accounts for the interactions of multiple target cells and their spatial and temporal relationships at each time frame. We condition our generative model on two starting frames and utilize an auto-regressive framework to predict the subsequent five frames. Our model achieves a 0.88 F1 score on the cell death event classification and 2.11 mean absolute death-time error, significantly outperforming state-of-the-art methods.

cell biology↗

bioRxiv2023

Overcoming migratory exhaustion: Expression of Matrix metalloproteinase-8 (MMP-8) promotes tumor infiltration and anti-tumor activity of CAR-T cells

Despite the encouraging success of chimeric antigen receptor (CAR) T-cell therapy in treating hematological malignancies, the translation of adoptive cell therapies to solid tumors remains a challenge. Several studies have attributed the inability of tumor-infiltrating T cells to traffic to solid tumors, primarily to the presence of the extracellular matrix (ECM) and immunosuppressive environment of solid tumors. The ability of the transferred T cells to infiltrate the tumor is an essential prerequisite for anti-tumor activity. We show here that upon activation and expansion, T cells quickly lose their migratory capacity, leading to migratory exhaustion. At the molecular level, migratory exhaustion could be attributed to the downregulation of matrix metalloproteinase 8 (MMP8). To overcome this, we hypothesized that T cells genetically modified to secrete the mature form of matrix metalloproteinase 8 (mMMP8) would facilitate migration across matrix barriers in vitro and in vivo. We demonstrated that CAR T cells that co-express mMMP8 demonstrate robust migration across Matrigel and can kill tumor cells embedded in Matrigel in vitro. We tested the efficacy of these mMMP8 engineered cells in both leukemic and ovarian cancer cell models embedded in Matrigel in xenograft mouse models. Our results illustrate that unlike parental CAR T cells that have minimal anti-tumor efficacy in these models, CAR T cells that secrete mMMP8 promote T-cell infiltration, leading to the eradication of the tumors and survival. We anticipate that the co-expression of mMMP8 can be broadly utilized to improve the infiltration and efficacy of CAR T cells targeting many different antigens.

cancer biology↗

bioRxiv2022

Engineering non-cytotoxic delivery of proteins by T cells via fusion to NPC2

Engineering cellular therapeutics by programming T cells has great potential in immunology. The primary mechanism employed by T cells for the specific transfer of proteins at the immunological synapse is via the lysosomal perforin pathway that facilitates the transfer of cytotoxic granzymes leading to apoptosis in target cells. Facilitating the delivery of non-cytotoxic proteins through perforin oligomers will dramatically expand the range of protein cargos that T cells can traffic to the target cells. Here, we have identified the intralysosomal protein, NPC2, as a chaperone that can facilitate the delivery of T-cell derived reporter proteins through perforin pores at the immunological synapse. Structural and biophysical considerations suggested that NPC2 could traverse through perforin pores and in vitro experiments confirmed the transport of purified NPC2 through perforin pores on cell membranes. To characterize the ability of NPC2 to facilitate the transfer of payloads in T cells, we constructed NPC2-mCherry fusion proteins in T cells. Using confocal microscopy and flow cytometry, we confirmed the colocalization of the NPC2 fused protein with lytic granules and the transfer of the fluorescent protein payload from T cells to target cells in co-culture experiments. The NPC2 fusion enabled the localization of mCherry to secretory lysosomes in mouse TCR CD8+ T cells and human CD4+ and CD8+ chimeric antigen receptor (CAR) T cells. These results illustrate that by using NPC2 as a molecular chaperone, the NPC2-perforin pathway can be exploited as a programmable molecular delivery system for cell-based therapies.

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↗