Kayla F. Goliwas

Novel preclinical models that better mimic the in vivo tumor microenvironment are essential to advance understanding of tumor biology and resistance/response to therapy. Herein, we report development of a novel ex vivo patient-derived three-dimensional lung tumor model (3D-LTM) for use in evaluating response to therapy. With this model system that maintains cell-cell interactions and tissue architecture, we observed heterogeneity of response to immune checkpoint inhibitors (ICI), as noted in non-small cell lung cancer (NSCLC) patients, and defined gene signatures associated with response. Spatial transcriptomics identified positive correlation of CD8⁺ T cell populations, CD4⁺ memory T cells, mast cells, NK cells, naive B cells, endothelial cells and non-classical monocytes with response status, and negative correlation of macrophages with response status. Pathway analysis of gene expression showed that chemokine signaling related pathways were activated in responder 3D-LTM tissues, whereas suppression of antigen presentation-related pathways and activation of T_reg differentiation-related pathways were associated with non-responder 3D-LTM tissues. Additionally, the abundance of dividing T cells and naive CD8⁺ T cells differentially correlated with T cell cytotoxicity gene signatures based on response status. Thus, this model may provide utility for rapid testing of therapeutic outcomes and biomarker development.

Triple-negative breast cancer (TNBC) is an aggressive subtype with limited treatment options. Emerging evidence shows that mechanotransduction, driven by matrix stiffness and mechanical signaling, promotes TNBC invasion and metastasis. As breast cancer progresses, expansion of fibroblasts and tumor-reactive stroma increases extracellular matrix deposition, generating matrix tension and enhancing mechanotransduction, which promotes cell proliferation, invasion, and metastatic potential through altered gene expression patterns. To investigate the molecular mechanisms underlying these changes, human TNBC cells were subjected to constant or oscillatory strain, followed by comprehensive transcriptomic analysis. Results revealed pronounced differential expression of genes involved in cell migration, adhesion, and transforming growth factor-β (TGFβ) signaling, with RT-PCR validation confirming SKI Like Proto Oncogene (SKIL) as the most strongly upregulated gene. Analysis of The Cancer Genome Atlas (TCGA) datasets indicated that SKIL is highly expressed in multiple breast cancer subtypes. Cross-sectional comparison of oscillatory strain-induced genes with TCGA data revealed coordinated upregulation of TGFβ, SKIL, and other genes associated with invasive phenotypes, immune suppression, and drug resistance, highlighting the vital role of TGFβ signaling. Transcription factor enrichment analysis further identified regulators linked to oncogenic pathways, including TGFβ effectors and Hippo signaling, supporting a mechanotransduction-driven transcriptional program in breast cancer.

Introduction: Multi-specific and long-lasting T-cell immunity has been recognized to indicate long-term protection against pathogens, including the novel coronavirus, SARS-CoV-2, which is the causative agent of the COVID-19 pandemic. Functional significance of peripheral memory T cells in individuals recovered from COVID-19 (COVID-19⁺) is beginning to be appreciated; however, the role of lung tissue-resident memory (lung TRM) T cells in SARS-CoV-2 infection is still being investigated. This is, in part, due to the lack of preclinical tissue models available to follow the convalescence period.Methods: Here, we utilize a perfused three-dimensional (3D) human lung-tissue model and show pre-existing local T-cell immunity against SARS-CoV-2 proteins in lung tissues.Results: We report ex vivo maintenance of functional multi-specific IFN-γ-secreting lung TRM T cells in COVID-19⁺ and their induction in lung tissues of vaccinated COVID-19⁺ subjects. Importantly, we identify SARS-CoV-2 peptide-responding memory B cells and IgA⁺ plasma cells in ex vivo cultured lung tissues of COVID-19⁺. Furthermore, lung tissue IgA levels were increased in COVID-19⁺ and responded to peptide stimulation.Discussion: In our study, we highlight the importance of utilization of human lung-tissue models to understand the local antiviral immune response in the lung to protect against SARS-CoV-2 infection.

Novel preclinical models that better mimic the in vivo tumor microenvironment are needed for advanced understanding of tumor biology and resistance/response to therapy. Herein, we report development of a novel ex vivo patient-derived three-dimensional lung tumor model (3D-LTM), that maintains features of human extracellular matrix, cell-cell interactions, and tissue architecture to evaluate a rapid response to immune checkpoint inhibitors (ICI). Within this model system, we recapitulated the heterogeneity of response to immunotherapy observed in non-small cell lung cancer (NSCLC) patients and defined signatures associated with response for predicting early response of ICI in patients. Spatial transcriptomics of the 3D-LTMs identified positive correlation of CD8 ⁺ T cell populations, CD4 ⁺ memory T cells, mast cells, NK cells, endothelial cells and non-classical monocytes with response status, whereas macrophages negatively correlated with response status. Pathway analysis of gene expression showed that chemokine signaling related pathways were activated in responder 3D-LTM tissues, whereas suppression of antigen presentation-related pathways and activation of T _reg differentiation-related pathways was associated with 3D-LTMs that were not considered responders. This model system has utility for rapid testing of novel immune directed therapy outcomes and for developing biomarkers of ICI response in NSCLC.

Hidradenitis suppurativa (HS) is a chronic, debilitating inflammatory skin disease with a poorly understood immunopathogenesis. Here, we report that HS lesional skin is characterized by the expansion of innate lymphocytes and T cells expressing CD2, an essential activation receptor and adhesion molecule. Lymphocytes expressing elevated CD2 predominated with unique spatial distribution throughout the epidermis and hypodermis in the HS lesion. CD2 ⁺ cells were mainly innate lymphocytes expressing the NK cell marker, CD56, and CD4 ⁺ T cells. Importantly, these CD2 ⁺ cells interacted with CD58 (LFA3) expressing epidermal keratinocytes and fibroblasts in the hypodermis. Granzyme A ^bright NKT cells (CD2 ⁺ CD3 ⁺ CD56 ^bright ) clustered with α-SMA expressing fibroblasts juxtaposed to epithelialized tunnels and fibrotic regions of the hypodermis. Whereas NK cells (CD2 ⁺ CD56 ^dim ) were perforin ⁺ , granzymes A ⁺ and B ⁺ , and enriched adjacent to hyperplastic follicular epidermis and tunnels of HS showing presence of apoptotic cells. The cytokines IL-12, IL-15, and IL-18, which enhance NK cell maturation and function were significantly elevated in HS. Ex vivo HS skin explant cultures treated with CD2:CD58 interaction-blocking anti-CD2 monoclonal antibody attenuated secretion of inflammatory cytokines/chemokines and suppressed inflammatory gene signature. Additionally, CD2:CD58 blockade altered miRNAs involved in NK/NKT differentiation and/or function. In summary, we show that a cellular network of heterogenous NKT and NK cell populations drives inflammation and is critical in the pathobiology of HS, including tunnel formation and fibrosis. Finally, CD2 blockade is a viable immunotherapeutic approach for the effective management of HS.

Exposure to heavy metals (HMs) is often associated with inflammation and cell death, exacerbating respiratory diseases including asthma. Most inhaled particulate HM exposures result in the deposition of HM-bound fine particulate matter, PM_2.5, in pulmonary cell populations. While localized high concentrations of HMs may be a causative factor, existing studies have mostly evaluated the effects of systemic or low-dose chronic HM exposures. This report investigates the impact of local high concentrations of specific HMs (NaAsO₂, MnCl₂, and CdCl₂) on sphingolipid homeostasis and oxidative stress, as both play a role in mediating responses to HM exposure and have been implicated in asthma. Utilizing an in vitro model system and three-dimensional ex vivo human tissue models, we evaluated the expression of enzymatic regulators of the salvage, recycling, and de novo synthesis pathways of sphingolipid metabolism, and observed differential modulation in these enzymes between HM exposures. Sphingolipidomic analyses of specific HM-exposed cells showed increased levels of anti-apoptotic sphingolipids and reduced pro-apoptotic sphingolipids, suggesting activation of the salvage and de novo synthesis pathways. Differential sphingolipid regulation was observed within HM-exposed lung tissues, with CdCl₂ exposure and NaAsO₂ exposure activating the salvage and de novo synthesis pathway, respectively. Additionally, using spatial transcriptomics and quantitative real-time PCR, we identified HM exposure-induced transcriptomic signatures of oxidative stress in epithelial cells and human lung tissues.

Hidradenitis suppurativa (HS) is a chronic debilitating inflammatory skin disease with poorly understood pathogenesis. Single-cell RNAseq analysis of HS lesional and healthy individual skins revealed that NKT and NK cell populations were greatly expanded in HS, and they expressed elevated CD2, an activation receptor. Immunohistochemistry analyses confirmed significantly expanded numbers of CD2+ cells distributed throughout HS lesional tissue, and many co-expressed the NK marker, CD56. While CD4+ T cells were expanded in HS, CD8 T cells were rare. CD20+ B cells in HS were localized within tertiary follicle like structures. Immunofluorescence microscopy showed that NK cells (CD2 ⁺ CD56 ^dim ) expressing perforin, granzymes A and B were enriched within the hyperplastic follicular epidermis and tunnels of HS and juxtaposed with apoptotic cells. In contrast, NKT cells (CD2 ⁺ CD3 ⁺ CD56 ^bright ) primarily expressed granzyme A and were associated with α-SMA expressing fibroblasts within the fibrotic regions of the hypodermis. Keratinocytes and fibroblasts expressed high levels of CD58 (CD2 ligand) and they interacted with CD2 expressing NKT and NK cells. The NKT/NK maturation and activating cytokines, IL-12, IL-15 and IL-18, were significantly elevated in HS. Inhibition of cognate CD2-CD58 interaction with blocking anti-CD2 mAb in HS skin organotypic cultures resulted in a profound reduction of the inflammatory gene signature and secretion of inflammatory cytokines and chemokines in the culture supernate. In summary, we show that a cellular network of heterogenous NKT and NK cell populations drives inflammation, tunnel formation and fibrosis in the pathogenesis of HS. Furthermore, CD2 blockade is a viable immunotherapeutic approach for the management of HS.

Cancer-associated fibroblasts (CAFs) have distinct roles within the tumor microenvironment, which can impact the mode and efficacy of tumor cell migration. CAFs are known to increase invasion of less-aggressive breast cancer cells through matrix remodeling and leader–follower dynamics. Here, we demonstrate that CAFs communicate with breast cancer cells through the formation of contact-dependent tunneling nanotubes (TNTs), which allow for the exchange of cargo between cell types. CAF mitochondria are an integral cargo component and are sufficient to increase the 3D migration of cancer cells. This cargo transfer results in an increase in mitochondrial ATP production in cancer cells, whereas it has a negligible impact on glycolytic ATP production. Manually increasing mitochondrial oxidative phosphorylation (OXPHOS) by providing extra substrates for OXPHOS fails to enhance cancer cell migration unless glycolysis is maintained at a constant level. Together, these data indicate that tumor–stromal cell crosstalk via TNTs and the associated metabolic symbiosis is a finely controlled mechanism by which tumor cells co-opt their microenvironment to promote cancer progression and may become a potential therapeutic target.

SUMMARY: Multi-specific and long-lasting T cell immunity have been recognized as indicators for long term protection against pathogens including the novel coronavirus SARS-CoV-2, the causative agent of the COVID-19 pandemic. Functional significance of peripheral memory T cells in individuals recovering from COVID-19 (COVID-19 ⁺ ) are beginning to be appreciated; but little is known about lung resident memory T cells (lung TRM) in SARS-CoV-2 infection. Here, we utilize a perfused three dimensional (3D) human lung tissue model and identify pre-existing local T cell immunity against SARS-CoV-2 proteins in lung tissues. We report ex vivo maintenance of functional multi-specific IFN-γ secreting lung TRM in COVID-19 ⁺ and their induction in lung tissues of vaccinated COVID-19 ⁺ . Importantly, we identify SARS-CoV-2 peptide-responding B cells and IgA ⁺ plasma cells in lung tissues of COVID-19 ⁺ in ex vivo 3D-tissue models. Our study highlights the importance of balanced and local anti-viral immune response in the lung with persistent induction of TRM and IgA ⁺ plasma cells for future protection against SARS-CoV-2 infection. Further, our data suggest that inclusion of multiple viral antigens in vaccine approaches may broaden the functional profile of memory T cells to combat the severity of coronavirus infection.