John Andrew Ronald

Pannexin 1 (PANX1) is a channel-forming glycoprotein that has been implicated in regulating melanoma cell properties such as growth, migration, and invasion. Here we report that the CRISPR-Cas9 mediated ablation of PANX1 or channel inhibition with probenecid shifted A375-P cell cycle distribution towards G2/M, while channel inhibition with spironolactone stalled cell cycle progression at the G1/S checkpoint regardless of PANX1 expression. Bulk RNA sequencing revealed cell cycle related gene expression was downregulated with PANX1 targeting strategies and this was exacerbated when PANX1 deficient cells were treated with spironolactone. When grown as spheroids, we found the deletion and inhibition of PANX1 disrupted spheroid compaction, with spheroids displaying poorly defined borders and reduced circularity. The deletion of PANX1 increased spheroid fragmentation, and this phenotype was intensified when spheroids devoid of PANX1 were treated with spironolactone, highlighting a potential synthetic lethal effect. Using liquid chromatography mass spectrometry, we sought to determine whether PANX1 had binding partners that may act to stabilize spheroid integrity and found that PANX1 co- immunoprecipitated various cell adhesion molecules such as β-actin and F-actin-capping protein subunit α-1, suggesting PANX1 and its interacting partners may stabilize cell-cell contacts. When control and PANX1 ablated cells were grown in chick chorioallantoic membrane xenografts to assess their tumour growth characteristics, we discovered that the deletion of PANX1 reduced tumour surface area and viability. Our findings indicate that PANX1 regulates cell cycle progression and the structural architecture of A375-P melanoma tumours, highlighting the therapeutic utility of targeting PANX1 in melanoma.

Cellular therapies harnessing the potential of endothelial colony-forming cells (ECFCs) for therapeutic revascularization in patients with critical limb ischemia (CLI) have garnered much interest. However, limited cell persistence following intramuscular injection into ischemic tissues has prompted the development of biomaterials as cell-delivery platforms to support cell survival and enhance therapeutic outcomes. This study explored the use of hydrogels derived from human decellularized adipose tissue (DAT) as a cell-instructive platform for ECFC delivery. In vitro studies confirmed high initial ECFC viability at 1 day following encapsulation in the DAT hydrogels but revealed that cell viability and proliferation were reduced over time in culture as compared to ECFCs cultured on tissue culture polystyrene (TCP). However, the ECFCs cultured within the DAT hydrogels showed similar ECFC cell surface marker expression patterns compared to ECFCs cultured on TCP after 6 days in culture, supporting that their phenotype was maintained. Subsequent testing focused on comparing a low (2.4 × 10⁵ cells) and high (1 × 10⁶ cells) dose of ECFCs delivered in either saline or within DAT hydrogels in a femoral artery ligation-induced CLI (FAL-CLI) model in NOD/SCID mice over 35 days. Bioluminescence imaging results showed that the low dose of ECFCs delivered in DAT hydrogels demonstrated enhanced retention on days 14, 21, and 28 compared to delivery in saline. Despite this finding, there were no observed improvements in hindlimb perfusion between delivery strategies, which may be related to the robust collateral vessel formation observed in this model with a µCT-based angiography method. Overall, this work supports that DAT hydrogels can enhance localized ECFC retention following intramuscular injection in mice with femoral artery ligation but emphasizes that enhanced retention may be insufficient for improving functional vascular regeneration.

Immune activity within tumors and secondary lymphoid organs critically influences cancer progression, metastasis, and treatment response. We present a dual-nucleus (¹H/¹⁹F) molecular MRI platform for non-invasive, high-resolution profiling of the tumor-immune microenvironment in immunocompetent mouse models. Tumor viability was visualized by engineering breast cancer cells to express a novel mouse-derived ¹H MRI reporter gene, enabling in vivo differentiation of viable and necrotic tumor regions. Concurrently, ¹⁹F MRI using perfluorocarbon (PFC) nanoemulsions enabled longitudinal tracking of immune cell infiltration, extending beyond conventional tumor-associated macrophage-focused approaches. Ex vivo analyses confirmed PFC uptake across diverse immune subsets, with tissue- and context-specific variations in ¹⁹F signal driven by differences in cell abundance and labeling efficiency. Notably, ¹⁹F MRI revealed a predominantly myeloid signature within tumors, a mixed myeloid/lymphoid profile in the spleen, and a lymphoid-skewed signal in tumor-draining lymph nodes. By integrating tumor-specific ¹H imaging with immune-resolving ¹⁹F imaging, this single-modality platform offers a comprehensive view of tumor architecture and immune cell presence within tumors. Our imaging approach enables discrimination of immunologically dense tumors and offers critical insights into the interpretation of ¹⁹F MRI signals within immune competent animal models, providing an immunoimaging tool that increases the translational relevance of preclinical therapeutic insights.

CRISPR-based genome editing therapeutics are entering the clinic, offering transformative potential but also presenting potential risks. Preclinical-to-clinical toolkits are needed to assess the safety and efficacy of these new therapies and accelerate progress. Emerging technologies to monitor the biological effects of genome editors cover a range of biological scales, from the direct measurement of editing outcomes in DNA, to human microphysiological systems, and non-invasive in vivo imaging. Measurements of on-target and off-target editing outcomes, including sequences unique to humans, provide essential benchmarks to understand functional responses. Microphysiological systems, including organoids and organs-on-chips, enable phenotypic evaluations of editing strategies in varied organ lineages and disease states. Non-invasive imaging modalities can track the biodistribution and activities of genome editors and edited cells in vivo. Collectively, these technologies provide complementary insights across different scales, from the single nucleotide to the whole organism, bridging preclinical therapeutics development with clinical trials.

Rationale: Chimeric 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 CAR-T cell potency and safety, yet challenges such as inefficient tumor homing and toxicities in normal tissues, limit broader adoption. Advanced imaging technologies, including bioluminescence imaging (BLI) and positron emission tomography (PET), provide real-time visualization of CAR-T cell behavior in vivo. 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.

Supporting Information for Manuscript

Metastasis is the leading cause of cancer-related death. However, it remains a poorly understood aspect of cancer biology, and most preclinical cancer studies do not examine metastasis, focusing solely on the primary tumor. One major factor contributing to this paradox is a gap in available tools for accurate spatiotemporal measurements of metastatic spread in vivo. Here, our objective was to develop an imaging reporter system that offers sensitive three-dimensional (3D) detection of cancer cells at high resolutions in live mice. An organic anion-transporting polypeptide 1b3 (oatp1b3) was used as an MRI reporter gene, and its sensitivity was systematically optimized for in vivo tracking of viable cancer cells in a spontaneous metastasis model. Metastases with oatp1b3-MRI could be observed at the single lymph node level and tracked over time as cancer cells spread to multiple lymph nodes and different organ systems in individual animals. While initial single lesions were successfully imaged in parallel via bioluminescence, later metastases were largely obscured by light scatter from the initial node. Importantly, MRI could detect micrometastases in lung tissue comprised on the order of 1,000 cancer cells. In summary, oatp1b3-MRI enables longitudinal tracking of cancer cells with combined high resolution and high sensitivity that provides 3D spatial information and the surrounding anatomical context.Significance:An MRI reporter gene system optimized for tracking metastasis in deep tissues at high resolutions and able to detect spontaneous micrometastases in lungs of mice provides a useful tool for metastasis research.

Cell therapies harnessing the pro-vascular regenerative capacities of mesenchymal stromal cell (MSC) populations, including human adipose-derived stromal cells (hASCs), have generated considerable interest as an emerging treatment strategy for peripheral arterial disease (PAD) and its progression to critical limb ischemia (CLI). There is evidence to support that polysaccharide hydrogels can enhance therapeutic efficacy when applied as minimally-invasive delivery systems to support MSC survival and retention within ischemic tissues. However, there has been limited research to date on the effects of hydrogel composition on the phenotype and function of encapsulated cell populations. Recognizing this knowledge gap, this study compared the pro-angiogenic function of hASCs encapsulated in distinct but similarly-modified natural polysaccharide hydrogels composed of methacrylated glycol chitosan (MGC) and methacrylated hyaluronic acid (MHA). Initial in vitro studies confirmed high viability (>85%) of the hASCs following encapsulation and culture in the MGC and MHA hydrogels over 14 days, with a decrease in the cell density observed over time. Moreover, higher levels of a variety of secreted pro-angiogenic and immunomodulatory factors were detected in conditioned media samples collected from the hASCs encapsulated in the MGC-based hydrogels compared to the MHA hydrogels. Subsequent testing focused on comparing hASC delivery within the MGC and MHA hydrogels to saline controls in a femoral artery ligation-induced CLI (FAL-CLI) model in athymic nu/nu mice over 28 days. For the in vivo studies, the hASCs were engineered to express tdTomato and firefly luciferase to quantitatively compare the efficacy of the two platforms in supporting the localized retention of viable ASCs through longitudinal cell tracking with bioluminescence imaging (BLI). Interestingly, hASC retention was significantly enhanced when the cells were delivered in the MHA hydrogels as compared to the MGC hydrogels or saline. However, laser Doppler perfusion imaging (LDPI) indicated that the restoration of hindlimb perfusion was similar between the treatment groups and controls. These findings were corroborated by endpoint immunofluorescence (IF) staining showing similar levels of CD31 ⁺ cells in the ligated limbs at 28 days in all groups. Overall, this study demonstrates that enhanced MSC retention may be insufficient to augment vascular regeneration, emphasizing the complexity of designing biomaterials platforms for MSC delivery for therapeutic angiogenesis. In addition, the data points to a potential challenge in approaches that seek to harness the paracrine functionality of MSCs, as strategies that increase the secretion of immunomodulatory factors that can aid in regeneration may also lead to more rapid MSC clearance in vivo .