Biology subjects

Sanchez-Pupo, R. E.

Publications and source records attributed to Sanchez-Pupo, R. E..

4 recordsLinked to original sources

bioRxiv2025

Imaging CRISPR-Edited CAR-T Cell Therapies with Optical and Positron Emission Tomography Reporters

RationaleChimeric antigen receptor (CAR) T cell therapies have shown remarkable success in treating hematological cancers and are increasingly demonstrating potential for solid tumors. CRISPR-based genome editing offers a promising approach to enhance the potency and safety of CAR-T cells. However, several challenges persist, including inefficient tumor homing and treatment-related toxicities in normal tissues, which continue to hinder widespread adoption. Advanced imaging technologies, including bioluminescence imaging (BLI) and positron emission tomography (PET), provide real-time insights into CAR-T cell distribution and activity in vivo, both in preclinical models and in patients. Here, we developed Trackable Reporter Adaptable CRISPR-Edited CAR (tRACE-CAR) T cells, a modular system for site-specific integration of CARs and imaging reporters. MethodsThe luciferase reporter AkaLuciferase (AkaLuc) or the human sodium iodide symporter (NIS) were cloned downstream of the CAR in adeno-associated virus (AAV) donors for BLI or PET tracking, respectively. CARs with imaging reporters were knocked into the TRAC locus of primary human T cells via CRISPR editing and AAV transduction. Editing efficiency was evaluated by flow cytometry and junction PCR. In vitro cytotoxicity was assessed by BLI using firefly luciferase (Fluc)-expressing cancer cells co-cultured with CAR-T cells at varying effector-to-target ratios. In vivo, BLI and PET imaging assessed CAR-AkaLuc and CAR-NIS T cell expansion and trafficking in Nod-SCID-gamma mice bearing xenograft tumors. ResultsT cell receptor (TCR) knockout efficiency exceeded 85%, with CAR expression observed in 70-80% of cells, depending on the reporter used. Reporter-engineered CAR-T cells retained functionality in vitro and exhibited significant cytotoxicity against target cancer cells, outperforming naive T cells. In vivo, AkaLuc BLI and 18F-tetrafluoroborate PET enabled non-invasive tracking of viable CAR-T cells. Notably, the route of administration (intravenous, peritumoral, or intraperitoneal) significantly influenced the distribution of CAR-T cells and their therapeutic effectiveness. ConclusiontRACE-CAR enabled precise optical and PET tracking of CAR-T cells in models of B cell leukemia and ovarian cancer, allowing dynamic, non-invasive monitoring of cell distribution in both tumors and off-target tissues. This imaging platform could lead to more personalized, effective CRISPR-edited CAR cell therapies.

immunology↗

bioRxiv2023

Global Pannexin 1-deletion increases tumor-infiltrating lymphocytes in the BRAF/Pten mouse melanoma model.

Immunotherapies for malignant melanoma seek to boost the anti-tumoral response of CD8+ T cells but have a limited patient response rate, in part due to limited tumoral immune cell infiltration. Genetic or pharmacological inhibition of Pannexin 1 (PANX1) channel-forming protein is known to decrease melanoma cell tumorigenic properties in vitro and ex vivo. Here, we crossed Panx1 knockout (Panx1-/-) mice with the inducible melanoma model: BrafCA, PtenloxP, Tyr::CreERT2 (BPC). We found that deleting the Panx1 gene in mice does not reduce BRAF(V600E)/Pten-driven primary tumor formation or improve survival. However, BPC-Panx1-/- mice tumors exhibited a significant increase in infiltration of CD8+ T lymphocytes with no changes in the expression of early T cell activation marker CD69, LAG-3 checkpoint receptor or PD-L1 in tumors when compared to BPC-Panx1+/+ genotype. Our results suggest that although a Panx1 deletion does not overturn the aggressive BRAF/Pten-driven melanoma progression in vivo, it does increase the infiltration of effector immune T cell populations in the tumor microenvironment. We propose that PANX1-targeted therapy could be explored as a strategy to increase tumor-infiltrating lymphocytes to boost anti-tumor immunity.

cancer biology↗

bioRxiv2022

Pannexin 3 channels regulate architecture, adhesion, barrier function and inflammation in the skin

The channel-forming glycoprotein Pannexin 3 (PANX3) functions in cutaneous wound healing and keratinocyte differentiation, but its role in skin homeostasis through aging is not yet understood. We found that PANX3 is absent in newborn skin but becomes upregulated with age. We characterized the skin of global Panx3 knockout mice (KO) and found that KO dorsal skin showed sex-differences at different ages, but generally had reduced dermal and hypodermal areas compared to aged-matched controls. Transcriptomic analysis of KO epidermis revealed reduced E-cadherin stabilization and Wnt signaling compared to WT, consistent with the inability of primary KO keratinocytes to adhere in culture, and diminished epidermal barrier function in KO mice. We also observed increased inflammatory signaling in KO epidermis and higher incidence of dermatitis in aged KO mice compared to wildtype controls. These findings suggest that during skin aging, PANX3 is critical in the maintenance of dorsal skin architecture, keratinocyte cell-cell and cell-matrix adhesion and inflammatory skin responses.

cell biology↗

bioRxiv2021

Pannexin 3 deletion reduces fat accumulation and inflammation in a sex-specific manner

BackgroundPannexin 3 (PANX3), is a channel-forming glycoprotein that enables nutrient-induced inflammation in vitro, and genetic linkage data suggests it regulates body mass index. Here, we characterized inflammatory and metabolic parameters in global Panx3 knockout (KO) mice in the context of forced treadmill running (FEX) and high fat diet (HFD). MethodsC57BL/6N (WT) and KO mice were randomized to either a FEX running protocol or no running (SED) from 24 until 30 weeks of age. Body weight was measured biweekly, and body composition was measured at 24 and 30 weeks of age. Male WT and KO mice were fed a HFD from 12 - 28 weeks of age. Metabolic organs were analyzed for a panel of inflammatory markers and PANX3 expression. ResultsIn females there were no significant differences in body composition between genotypes, which could be due to the lack of PANX3 expression in female white adipose tissue, while male KOs fed a chow diet had lower body weight, and lower fat mass at 24 and 30 weeks of age, which was reduced to the same extent as 6 weeks of FEX in WT mice. Additionally, male KO mice exhibited significantly lower expression of multiple pro-inflammatory genes in white adipose tissue compared to WT mice. While on a HFD body weight differences were insignificant, in KO mice, multiple inflammatory genes were significantly differently expressed in quadriceps muscle and white adipose tissue resulting in a more anti-inflammatory phenotype compared to WT mice. The lower fat mass in male KO mice may be due to significantly fewer adipocytes in their subcutaneous fat compared to WT mice. Mechanistically, adipose stromal cells (ASCs) cultured from KO mice grow significantly slower than WT ASCs. ConclusionPANX3 is expressed in male adult mouse adipose tissue and may regulate adipocyte numbers, influencing fat accumulation and inflammation.

physiology↗