Induction of hemangiosarcoma in mice after chronic treatment with S1P-modulator siponimod and its lack of relevance to rat and human.

A high incidence of hemangiosarcoma (HSA) was observed in mice treated for 2 years with siponimod, a sphingosine-1-phosphate receptor 1 (S1P1) functional antagonist, while no such tumors were observed in rats under the same treatment conditions. In 3-month rat (90 mg/kg/day) and 9-month mouse (25 and 75 mg/kg/day) in vivo mechanistic studies, vascular endothelial cell (VEC) activation was observed in both species, but VEC proliferation and persistent increases in circulating placental growth factor 2 (PLGF2) were only seen in the mouse. In mice, these effects were sustained over the 9-month study duration, while in rats increased mitotic gene expression was present at day 3 only and PLGF2 was induced only during the first week of treatment. In the mouse, the persistent VEC activation, mitosis induction, and PLGF2 stimulation likely led to sustained neo-angiogenesis which over life-long treatment may result in HSA formation. In rats, despite sustained VEC activation, the transient mitotic and PLGF2 stimuli did not result in the formation of HSA. In vitro, the mouse and rat primary endothelial cell cultures mirrored their respective in vivo findings for cell proliferation and PLGF2 release. Human VECs, like rat cells, were unresponsive to siponimod treatment with no proliferative response and no release of PLGF2 at all tested concentrations. Hence, it is suggested that the human cells also reproduce a lack of in vivo response to siponimod. In conclusion, the molecular mechanisms leading to siponimod-induced HSA in mice are considered species specific and likely irrelevant to humans.

Sequential Chromogenic IHC: Spatial Analysis of Lymph Nodes Identifies Contact Interactions between Plasmacytoid Dendritic Cells and Plasmablasts.

Recent clinical observations have emphasized the critical role that the spatial organization of immune cells in lymphoid structures plays in the success of cancer immunotherapy and patient survival. However, implementing sequential chromogenic IHC (scIHC) to analyze multiple biomarkers on a single tissue section has been limited because of a lack of a standardized, rigorous guide to the development of customized biomarker panels and a need for user-friendly analysis pipelines that can extract meaningful data. In this context, we provide a comprehensive guide for the development of novel biomarker panels for scIHC, using practical examples and illustrations to highlight the most common complications that can arise during the setup of a new biomarker panel, and provide detailed instructions on how to prevent and detect cross-reactivity between secondary reagents and carryover between detection antibodies. We also developed a novel analysis pipeline based on non-rigid tissue deformation correction, Cellpose-inspired automated cell segmentation, and computational network masking of low-quality data. We applied this biomarker panel and pipeline to study regional lymph nodes from patients with head and neck cancer, identifying novel contact interactions between plasmablasts and plasmacytoid dendritic cells in vivo. Given that Toll-like receptors, which are highly expressed in plasmacytoid dendritic cells, play a key role in vaccine efficacy, the significance of this cell-cell interaction decisively warrants further studies. In summary, this work provides a streamlined approach to the development of customized biomarker panels for scIHC that will ultimately improve our understanding of immune responses in cancer. Significance: We present a comprehensive guide for developing customized biomarker panels to investigate cell-cell interactions in the context of immune responses in cancer. This approach revealed novel contact interactions between plasmablasts and plasmacytoid dendritic cells in lymph nodes from patients with head and neck cancer.

Cancer stem cell-derived extracellular vesicles preferentially target MHC-II-macrophages and PD1+ T cells in the tumor microenvironment.

Immunotherapy is an approved treatment option for head and neck squamous cell carcinoma (HNSCC). However, the response rate to immune checkpoint blockade is only 13% for recurrent HNSCC, highlighting the urgent need to better understand tumor-immune interplay, with the ultimate goal of improving patient outcomes. HNSCC present high local recurrence rates and therapy resistance that can be attributed to the presence of cancer stem cells (CSC) within tumors. CSC exhibit singular properties that enable them to avoid immune detection and eradication. How CSC communicate with immune cells and which immune cell types are preferentially found within the CSC niche are still open questions. Here, we used genetic approaches to specifically label CSC-derived extracellular vesicles (EVs) and to perform Sortase-mediated in vivo proximity labeling of CSC niche cells. We identified specific immune cell subsets that were selectively targeted by EVCSC and that were found in the CSC niche. Native EVCSC preferentially targeted MHC-II-macrophages and PD1+ T cells in the tumor microenvironment, which were the same immune cell subsets enriched within the CSC niche. These observations indicate that the use of genetic technologies able to track EVs without in vitro isolation are a valuable tool to unveil the biology of native EVCSC.

Characterization of the tumor immune microenvironment of sinonasal squamous-cell carcinoma.

BACKGROUND: Treatment and prognosis of sinonasal squamous-cell carcinoma (SNSCC) have not significantly improved despite improvements in radical therapy. Characterization of the tumor immune microenvironment (TiME) may identify patient subgroups associated with disease recurrence, and provide new biomarkers for improved patient stratification and treatment. METHODS: The TiME was quantitatively evaluated by multiplex immunohistochemistry (mIHC) in archived tissue sections from 38 patients with SNSCC, and were assessed for differences between recurrent (n = 20) and nonrecurrent (n = 18) groups. Hierarchical clustering analyses were performed to identify phenotypic TiME subgroups within the cohort and were used to compare survival outcomes. RESULTS: Our mIHC analysis revealed increased T-cell populations and decreased myeloid-cell populations in SNSCC patients without recurrent disease, as compared with patients with recurrent disease. Within T-cell subsets, there was a significantly higher percentage of granzyme B+ , T-bet+ , Eomes+ T cells, as well as higher proliferation of CD8+ T cells within the nonrecurrent group relative to the recurrent group. Furthermore, immune-cell complexity profiles of SNSCC revealed hyper- and hypo-T-cell-inflamed, myeloid-inflamed, B-cell-inflamed, and broadly hypoinflamed subtypes not previously identified by gene expression analyses. Our study revealed that presence of either hyper- or hypo-T-cell-inflamed TiME subtypes were associated with increased survival outcomes as compared with broadly hypoinflamed TiME subtypes (p = 0.035 and 0.0376, respectively). CONCLUSIONS: The TiME of SNSCC reveals distinct subtypes, which may correlate with recurrence and survival outcomes.

Gene Expression Profiling of Lymph Node Sub-Capsular Sinus Macrophages in Cancer.

Lymph nodes are key lymphoid organs collecting lymph fluid and migratory cells from the tissue area they survey. When cancerous cells arise within a tissue, the sentinel lymph node is the first immunological organ to mount an immune response. Sub-capsular sinus macrophages (SSMs) are specialized macrophages residing in the lymph nodes that play important roles as gatekeepers against particulate antigenic material. In the context of cancer, SSMs capture tumor-derived extracellular vesicles (tEVs), a form of particulate antigen released in high amounts by tumor cells. We and others have recently demonstrated that SSMs possess anti-tumor activity because in their absence tumors progress faster. A comprehensive profiling of SSMs represents an important first step to identify the cellular and molecular mechanisms responsible for SSM anti-tumor activity. Unfortunately, the isolation of SSMs for molecular analyses is very challenging. Here, we combined an optimized dissociation protocol, careful marker selection and stringent gating strategies to highly purify SSMs. We provide evidence of decreased T and B cell contamination, which allowed us to reveal the gene expression profile of this elusive macrophage subset. Squamous cell carcinomas induced an increase in the expression of Fc receptors, lysosomal and proteasomal enzymes in SSMs. Imaging of mouse and patient lymph nodes confirmed the presence of the top differentially expressed genes. These results suggest that SSMs respond to tumor formation by upregulating the machinery necessary for presentation of tumor particulate antigens to B cells.

Cell Surface Labeling by Engineered Extracellular Vesicles.

Extracellular vesicles (EVs) can mediate local and long-range intercellular communication via cell surface signaling. In order to perform in vivo studies of unmanipulated, endogenously released EVs, sensitive but stringent approaches able to detect EV-cell surface interactions are needed. However, isolation and reinfusion of EVs can introduce biases. A rigorous way to study EVs in vivo is by genetically engineering membrane-bound reporters into parental cells. Still, the amount of reporter molecules that EVs can carry is relatively small, and thus, the sensitivity of the approach is suboptimal. This work addresses this issue by engineering EVs to display a membrane-bound form of Sortase A (SrtA), a bacterial transpeptidase that can catalyze the transfer of reporter molecules on the much bigger surface of EV-binding cells. SrtA design and reaction requirements are optimized and validated. Efficient in vitro labeling of EV-binding cells is achieved, even in the presence of only one N-terminal glycine on cell surface proteins. As compared to indirect labeling of EV-binding cells (e.g., using CD63-GFP fusion), the SrtA-based approach shows 1-2 log increase in sensitivity, depending on the EV source. This novel approach will be useful to identify and study the full set of host cells interacting with native EVs in vivo.

Dynamic Treg interactions with intratumoral APCs promote local CTL dysfunction.

Tregs control various functions of effector T cells; however, where and how Tregs exert their immunomodulatory effects remain poorly understood. Here we developed a murine model of adoptive T cell therapy and found that Tregs induce a dysfunctional state in tumor-infiltrating CTLs that resembles T cell exhaustion and is characterized by low expression of effector cytokines, inefficient cytotoxic granule release, and coexpression of coinhibitory receptors PD-1 and TIM-3. Induction of CTL dysfunction was an active process, requiring local TCR signals in tumor tissue. Tregs infiltrated tumors only subsequent to Ag-dependent activation and expansion in tumor-draining LNs; however, Tregs also required local Ag reencounter within tumor tissue to induce CTL dysfunction and prevent tumor rejection. Multiphoton intravital microscopy revealed that in contrast to CTLs, Tregs only rarely and briefly interrupted their migration in tumor tissue in an Ag-dependent manner and formed unstable tethering-interactions with CD11c+ APCs, coinciding with a marked reduction of CD80 and CD86 on APCs. Activation of CTLs by Treg-conditioned CD80/86lo DCs promoted enhanced expression of both TIM-3 and PD-1. Based on these data, we propose that Tregs locally change the costimulatory landscape in tumor tissue through transient, Ag-dependent interactions with APCs, thus inducing CTL dysfunction by altering the balance of costimulatory and coinhibitory signals these cells receive.

PEG-like nanoprobes: multimodal, pharmacokinetically and optically tunable nanomaterials.

"PEG-like Nanoprobes" (PN's) are pharmacokinetically and optically tunable nanomaterials whose disposition in biological systems can be determined by fluorescence or radioactivity. PN's feature a unique design where a single PEG polymer surrounds a short fluorochrome and radiometal bearing peptide, and endows the resulting nanoprobe with pharmacokinetic control (based on molecular weight of the PEG selected) and optical tunability (based on the fluorochrome selected), while the chelate provides a radiolabeling option. PN's were used to image brain capillary angiography (intravital 2-photon microscopy), tumor capillary permeability (intravital fluorescent microscopy), and the tumor enhanced permeability and retention (EPR) effect (111In-PN and SPECT). Clinical applications of PN's include use as long blood half-life fluorochromes for intraoperative angiography, for measurements of capillary permeability in breast cancer lesions, and to image EPR by SPECT, for stratifying patient candidates for long-circulating nanomedicines that may utilize the EPR mechanism.