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Head and neck squamous cell carcinomas (HNSCCs) are the seventh most common cancer and represent a global health burden. Immune checkpoint inhibitors (ICIs) have shown promise in treating recurrent/metastatic disease with durable benefit in ∼30% of patients. Current biomarkers for HNSCC are limited in their dynamic ability to capture tumor microenvironment (TME) features with an increasing need for deeper tissue characterization. Therefore, new biomarkers are needed to accurately stratify patients and predict responses to therapy. Here, we have optimized and applied an ultra-high plex, single-cell spatial protein analysis in HNSCC. Tissues were analyzed with a panel of 101 antibodies that targeted biomarkers related to tumor immune, metabolic and stress microenvironments. Our data uncovered a high degree of intra-tumoral heterogeneity intrinsic to HNSCC and provided unique insights into the biology of the disease. In particular, a cellular neighborhood analysis revealed the presence of six unique spatial neighborhoods enriched in functionally specialized immune subsets. In addition, functional phenotyping based on key metabolic and stress markers identified four distinct tumor regions with differential protein signatures. One region was marked by infiltration of CD8+ cytotoxic T cells and overexpression of BAK, a proapoptotic regulator, suggesting strong immune activation and stress. Another adjacent region within the same tumor had high expression of G6PD and MMP9, known drivers of tumor resistance and invasion, respectively. This dichotomy of immune activation-induced death and tumor progression in the same sample demonstrates the heterogenous niches and competing microenvironments that may underpin variable clinical responses. Our data integrate single-cell ultra-high plex spatial information with the functional state of the TME to provide insights into HNSCC biology and differential responses to ICI therapy. We believe that the approach outlined in this study will pave the way toward a new understanding of TME features associated with response and sensitivity to ICI therapies.

A personal narrative is presented which explores the author's experience of visiting the convent of Carmel of the Holy Cross.

The anterior cruciate ligament has little capacity to heal after injury, in contrast to the medial collateral ligament. Cell migration and cell proliferation are essential steps in the wound healing of connective tissue, and we have developed an in vitro assay to study cell outgrowth (i.e., both cell migration and cell proliferation) from tissue explants. Using this assay, we have previously shown a synergistic effect of four growth factors on the early stages of cell outgrowth (i.e., at days 3 and 6) from these explants. Hyaluronan, a linear polysaccharide with a molecular weight of 3 × 106daltons, has been shown to enhance the anterior cruciate ligament healing capacity in vivo by over 50%. The purpose of this study was to assess in vitro whether hyaluronan and the combination of growth factors (transforming growth factor‐β1 25 ng/ml, platelet‐derived growth factor‐BB 100 ng/ml, basic fibroblast growth factor 100 ng/ml, and insulin 25 µg/ml) would enhance cell outgrowth from explants at 3 and 6 days. At 3 days the mean cell outgrowth from the explants treated with hyaluronan and the combination of growth factors exceeded the growth in the explants treated with growth factors alone for the anterior cruciate ligament (mean cell count ± standard error: 19 ± 4 and 2 ± 1 cells, respectively) and for the medial collateral ligament (297 ± 40 and 87 ± 18 cells, respectively). At 6 days, the mean cell outgrowth in the presence of growth factors was enhanced sixfold by hyaluronan for the anterior cruciate ligament (160 ± 27 compared with 26 ± 15 cells) and fourfold for the medial collateral ligament (1363 ± 219 compared with 330 ± 74 cells). As previously shown, little cell outgrowth occurred from the anterior cruciate or medial collateral ligament explants when no growth factors were added to the modified Eagle's media at 3 and 6 days (<20 cells). For the medial collateral ligament only, hyaluronan had a small stimulatory effect on cell outgrowth in minimal essential media in the absence of growth factors. At 3 days the stimulation by hyaluronan was fourfold (31 ± 8 cells compared with 7 ± 4 cells), and at 6 days the stimulation by hyaluronan was nearly fourfold (73 ± 17 compared with 20 ± 12 cells). These results suggest that hyaluronan could be used together with growth factors in efforts to enhance the healing of ligamentous tissue injuries.

Suggests a way for primary teachers to keep track of students' make-up work. Filling up of an assignment sheet for absent student.