NK and NKT cells in the pathogenesis of Hidradenitis suppurativa: Novel therapeutic strategy through targeting of CD2.

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.

Altered sphingolipid pathway in SARS-CoV-2 infected human lung tissue.

Introduction: The SARS-CoV-2 mediated COVID-19 pandemic has impacted millions worldwide. Hyper-inflammatory processes, including cytokine storm, contribute to long-standing tissue injury and damage in COVID-19. The metabolism of sphingolipids as regulators of cell survival, differentiation, and proliferation has been implicated in inflammatory signaling and cytokine responses. Sphingosine-kinase-1 (SK1) and ceramide-synthase-2 (CERS2) generate metabolites that regulate the anti- and pro-apoptotic processes, respectively. Alterations in SK1 and CERS2 expression may contribute to the inflammation and tissue damage during COVID-19. The central objective of this study is to evaluate structural changes in the lung post-SARS-CoV-2 infection and to investigate whether the sphingolipid rheostat is altered in response to SARS-CoV-2 infection. Methods: Central and peripheral lung tissues from COVID-19+ or control autopsies and resected lung tissue from COVID-19 convalescents were subjected to histologic evaluation of airspace and collagen deposisiton, and immunohistochemical evaluation of SK1 and CERS2. Results: Here, we report significant reduction in air space and increase in collagen deposition in lung autopsy tissues from patients who died from COVID-19 (COVID-19+) and COVID-19 convalescent individuals. SK1 expression increased in the lungs of COVID-19+ autopsies and COVID-19 convalescent lung tissue compared to controls and was mostly associated with Type II pneumocytes and alveolar macrophages. No significant difference in CERS2 expression was noted. SARS-CoV-2 infection upregulates SK1 and increases the ratio of SK1 to CERS2 expression in lung tissues of COVID-19 autopsies and COVID-19 convalescents. Discussion: These data suggest an alteration in the sphingolipid rheostat in lung tissue during COVID-19, suggesting a potential contribution to the inflammation and tissue damage associated with viral infection.

Mitochondrial transfer from cancer-associated fibroblasts increases migration in aggressive breast cancer.

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.

Ex Vivo Culture Models of Hidradenitis Suppurativa for Defining Molecular Pathogenesis and Treatment Efficacy of Novel Drugs.

Hidradenitis suppurativa (HS) is a complex and debilitating inflammatory skin disease for which no effective treatment is available currently. This is partly because of the lack of adequate human or animal models for defining the pathobiology of the disease. Here, we describe the development of air-liquid (A-L) interface, liquid-submersion (L-S), and bioreactor (Bio) ex vivo skin culture models. All three ex vivo platforms were effective for culturing skin samples for up to 14 days. Tissue architecture and integrity remained intact for at least 3 days for healthy skin and 14 days for HS skin. Up to day 3, no significant differences were observed in % early apoptotic cells among all three platforms. However, late apoptotic/necrotic cell death was increased in HS skin at day 3 in A-L and Bio culture. These cultures efficiently support the growth of various cells populations, including keratinocytes and immune cells. Profiling inflammatory gene signatures in HS skin from these ex vivo cultures showed dynamic changes in expression at day 3 and day 14. All three culture platforms were necessary to represent the inflammatory gene status of HS skin at day 0, suggesting that not all gene clusters were identically altered in each culture method. Similarly, cytokine/chemokine profiling of the supernatants from vehicle- and drug-treated ex vivo HS cultures again showed a better prediction of drug efficacy against HS. Overall, development of these three culture systems collectively provides a powerful tool to uncover the pathobiology of HS progression and screen various drugs against HS.

Local SARS-CoV-2 Peptide-Specific Immune Responses in Lungs of Convalescent and Uninfected Human Subjects.

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.

Sphingolipids in Lung Pathology in the Coronavirus Disease Era: A Review of Sphingolipid Involvement in the Pathogenesis of Lung Damage.

Sphingolipids are bioactive lipids involved in the regulation of cell survival, proliferation, and the inflammatory response. The SphK/S1P/S1PR pathway (S1P pathway) is a driver of many anti-apoptotic and proliferative processes. Pro-survival sphingolipid sphingosine-1-phosphate (S1P) initiates its signaling cascade by interacting with various sphingosine-1-phosphate receptors (S1PR) through which it is able to exert its pro-survival or inflammatory effects. Whereas sphingolipids, including ceramides and sphingosines are pro-apoptotic. The pro-apoptotic lipid, ceramide, can be produced de novo by ceramide synthases and converted to sphingosine by way of ceramidases. The balance of these antagonistic lipids and how this balance manifests is the essence of the sphingolipid rheostat. Recent studies on SARS-CoV-2 have implicated the S1P pathway in the pathogenesis of novel coronavirus disease COVID-19-related lung damage. Accumulating evidence indicates that an aberrant inflammatory process, known as "cytokine storm" causes lung injury in COVID-19, and studies have shown that the S1P pathway is involved in signaling this hyperinflammatory response. Beyond the influence of this pathway on cytokine storm, over the last decade the S1P pathway has been investigated for its role in a wide array of lung pathologies, including pulmonary fibrosis, pulmonary arterial hypertension (PAH), and lung cancer. Various studies have used S1P pathway modulators in models of lung disease; many of these efforts have yielded results that point to the potential efficacy of targeting this pathway for future treatment options. Additionally, they have emphasized S1P pathway's significant role in inflammation, fibrosis, and a number of other endothelial and epithelial changes that contribute to lung damage. This review summarizes the S1P pathway's involvement in COVID-19 and chronic lung diseases and discusses the potential for targeting S1P pathway as a therapeutic option for these diseases.

Extracellular Vesicle Mediated Tumor-Stromal Crosstalk Within an Engineered Lung Cancer Model.

Tumor-stromal interactions within the tumor microenvironment (TME) influence lung cancer progression and response to therapeutic interventions, yet traditional in vitro studies fail to replicate the complexity of these interactions. Herein, we developed three-dimensional (3D) lung tumor models that mimic the human TME and demonstrate tumor-stromal crosstalk mediated by extracellular vesicles (EVs). EVs released by tumor cells, independent of p53 status, and fibroblasts within the TME mediate immunomodulatory effects; specifically, monocyte/macrophage polarization to a tumor-promoting M2 phenotype within this 3D-TME. Additionally, immune checkpoint inhibition in a 3D model that included T cells showed an inhibition of tumor growth and reduced hypoxia within the TME. Thus, perfused 3D tumor models incorporating diverse cell types provide novel insights into EV-mediated tumor-immune interactions and immune-modulation for existing and emerging cancer therapies.

Moving Immune Therapy Forward Targeting TME.

The host immune system shapes the fate of tumor progression. Hence, manipulating patients' immune system to activate host immune responses against cancer pathogenesis is a promising strategy to develop effective therapeutic interventions for metastatic and drug-resistant cancers. Understanding the dynamic mechanisms within the tumor microenvironment (TME) that contribute to heterogeneity and metabolic plasticity is essential to enhance the patients' responsiveness to immune targeted therapies. Riera-Domingo et al. (Riera-Domingo C, Audige A, Granja S, Cheng WC, Ho PC, Baltazar F, Stockmann C, Mazzone, M. Physiol Rev 100: 1-102, 2020) describe the immune landscape within the TME and highlight the significance of metabolic and hypoxic signatures that impact immune function and response to immunotherapy strategies. Current literature in this field confirms that targeting tumor metabolism and the acidic microenvironment commonly associated with tumors may present viable strategies to modulate the host immune system in favor of response to immune targeted therapies. However, development of better tools to understand tumor-immune interactions and identify mechanisms driving nonresponders, more innovative clinical trial design, and new therapies will need to be identified to move the field forward. Personalized immune therapies incorporating metabolic and microbiome-based gene signatures to influence the therapeutic response and novel methods to generate immunologically "hot" tumors are at the forefront of immunotherapy currently. The combination of these approaches with clinically approved immunotherapies will be valuable moving forward.

Mechanical strain induces phenotypic changes in breast cancer cells and promotes immunosuppression in the tumor microenvironment.

Breast cancer (BCa) proliferates within a complex, three-dimensional microenvironment amid heterogeneous biochemical and biophysical cues. Understanding how mechanical forces within the tumor microenvironment (TME) regulate BCa phenotype is of great interest. We demonstrate that mechanical strain enhanced the proliferation and migration of both estrogen receptor+ and triple-negative (TNBC) human and mouse BCa cells. Furthermore, a critical role for exosomes derived from cells subjected to mechanical strain in these pro-tumorigenic effects was identified. Exosome production by TNBC cells increased upon exposure to oscillatory strain (OS), which correlated with elevated cell proliferation. Using a syngeneic, orthotopic mouse model of TNBC, we identified that preconditioning BCa cells with OS significantly increased tumor growth and myeloid-derived suppressor cells (MDSCs) and M2 macrophages in the TME. This pro-tumorigenic myeloid cell enrichment also correlated with a decrease in CD8+ T cells. An increase in PD-L1+ exosome release from BCa cells following OS supported additive T cell inhibitory functions in the TME. The role of exosomes in MDSC and M2 macrophage was confirmed in vivo by cytotracking fluorescent exosomes, derived from labeled 4T1.2 cells, preconditioned with OS. In addition, in vivo internalization and intratumoral localization of tumor-cell derived exosomes was observed within MDSCs, M2 macrophages, and CD45-negative cell populations following direct injection of fluorescently-labeled exosomes. Our data demonstrate that exposure to mechanical strain promotes invasive and pro-tumorigenic phenotypes in BCa cells, indicating that mechanical strain can impact the growth and proliferation of cancer cell, alter exosome production by BCa, and induce immunosuppression in the TME by dampening anti-tumor immunity.

Energetic regulation of coordinated leader-follower dynamics during collective invasion of breast cancer cells.

The ability of primary tumor cells to invade into adjacent tissues, followed by the formation of local or distant metastasis, is a lethal hallmark of cancer. Recently, locomoting clusters of tumor cells have been identified in numerous cancers and associated with increased invasiveness and metastatic potential. However, how the collective behaviors of cancer cells are coordinated and their contribution to cancer invasion remain unclear. Here we show that collective invasion of breast cancer cells is regulated by the energetic statuses of leader and follower cells. Using a combination of in vitro spheroid and ex vivo organoid invasion models, we found that cancer cells dynamically rearrange leader and follower positions during collective invasion. Cancer cells invade cooperatively in denser collagen matrices by accelerating leader-follower switching thus decreasing leader cell lifetime. Leader cells exhibit higher glucose uptake than follower cells. Moreover, their energy levels, as revealed by the intracellular ATP/ADP ratio, must exceed a threshold to invade. Forward invasion of the leader cell gradually depletes its available energy, eventually leading to leader-follower transition. Our computational model based on intracellular energy homeostasis successfully recapitulated the dependence of leader cell lifetime on collagen density. Experiments further supported model predictions that decreasing the cellular energy level by glucose starvation decreases leader cell lifetime whereas increasing the cellular energy level by AMP-activated kinase (AMPK) activation does the opposite. These findings highlight coordinated invasion and its metabolic regulation as potential therapeutic targets of cancer.

Methods to Evaluate Cell Growth, Viability, and Response to Treatment in a Tissue Engineered Breast Cancer Model.

The use of in vitro, engineered surrogates in the field of cancer research is of interest for studies involving mechanisms of growth and metastasis, and response to therapeutic intervention. While biomimetic surrogates better model human disease, their complex composition and dimensionality make them challenging to evaluate in a real-time manner. This feature has hindered the broad implementation of these models, particularly in drug discovery. Herein, several methods and approaches for the real-time, non-invasive analysis of cell growth and response to treatment in tissue-engineered, three-dimensional models of breast cancer are presented. The tissue-engineered surrogates used to demonstrate these methods consist of breast cancer epithelial cells and fibroblasts within a three dimensional volume of extracellular matrix and are continuously perfused with nutrients via a bioreactor system. Growth of the surrogates over time was measured using optical in vivo (IVIS) imaging. Morphologic changes in specific cell populations were evaluated by multi-photon confocal microscopy. Response of the surrogates to treatment with paclitaxel was measured by optical imaging and by analysis of lactate dehydrogenase and caspase-cleaved cytokeratin 18 in the perfused medium. Each method described can be repeatedly performed during culture, allowing for real-time, longitudinal analysis of cell populations within engineered tumor models.

Flow-perfusion bioreactor system for engineered breast cancer surrogates to be used in preclinical testing.

There is a need for preclinical testing systems that predict the efficacy, safety and pharmacokinetics of cancer therapies better than existing in vitro and in vivo animal models. An approach to the development of predictive in vitro systems is to more closely recapitulate the cellular and spatial complexity of human cancers. One limitation of using current in vitro systems to model cancers is the lack of an appropriately large volume to accommodate the development of this complexity over time. To address this limitation, we have designed and constructed a novel flow-perfusion bioreactor system that can support large-volume, engineered tissue comprised of multicellular cancer surrogates by modifying current microfluidic devices. Key features of this technology are a three-dimensional (3D) volume (1.2 cm3 ) that has greater tissue thickness than is utilized in existing microfluidic systems and the ability to perfuse the volume, enabling the development of realistic tumour geometry. The constructs were fabricated by infiltrating porous carbon foams with an extracellular matrix (ECM) hydrogel and engineering through-microchannels. The carbon foam structurally supported the hydrogel and microchannel patency for up to 161 h. The ECM hydrogel was shown to adhere to the carbon foam and polydimethylsiloxane flow chamber, which housed the hydrogel-foam construct, when surfaces were coated with glutaraldehyde (carbon foam) and nitric acid (polydimethylsiloxane). Additionally, the viability of breast cancer cells and fibroblasts was higher in the presence of perfused microchannels in comparison to similar preparations without microchannels or perfusion. Therefore, the flow-perfusion bioreactor system supports cell viability in volume and stromal contexts that are physiologically-relevant. Copyright © 2015 John Wiley & Sons, Ltd.

A recapitulative three-dimensional model of breast carcinoma requires perfusion for multi-week growth.

Breast carcinomas are complex, three-dimensional tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix. In vitro models that more faithfully recapitulate this dimensionality and stromal microenvironment should more accurately elucidate the processes driving carcinogenesis, tumor progression, and therapeutic response. Herein, novel in vitro breast carcinoma surrogates, distinguished by a relevant dimensionality and stromal microenvironment, are described and characterized. A perfusion bioreactor system was used to deliver medium to surrogates containing engineered microchannels and the effects of perfusion, medium composition, and the method of cell incorporation and density of initial cell seeding on the growth and morphology of surrogates were assessed. Perfused surrogates demonstrated significantly greater cell density and proliferation and were more histologically recapitulative of human breast carcinoma than surrogates maintained without perfusion. Although other parameters of the surrogate system, such as medium composition and cell seeding density, affected cell growth, perfusion was the most influential parameter.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates.

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

The presence of primary cilia in cancer cells does not predict responsiveness to modulation of smoothened activity.

Primary cilia are microtubule-based organelles that regulate smoothened-dependent activation of the GLI transcription factors in canonical hedgehog signaling. In many cancers, primary cilia are markedly decreased or absent. The lack of primary cilia may inhibit or alter canonical hedgehog signaling and, thereby, interfere in the cellular responsiveness to modulators of smoothened activity. Clinical trials of smoothened antagonists for cancer treatment have shown the best response in basal cell carcinomas, with limited response in other solid tumors. To determine whether the presence or absence of primary cilia in cancer cells will predict their responsiveness to modulation of smoothened activity, we compared the ability of an agonist and/or inhibitor of smoothened (SAG and SANT1, respectively) to modulate GLI-mediated transcription, as measured by GLI1 mRNA level or GLI-luciferase reporter activity, in non-cancer cells with primary cilia (ovarian surface epithelial cells and breast fibroblasts), in cancer cells that cannot assemble primary cilia (MCF7, MDA-MB-231 cell lines), and in cancer cells with primary cilia (SKOV3, PANC1 cell lines). As expected, SAG and SANT1 resulted in appropriate modulation of GLI transcriptional activity in ciliated non-cancer cells, and failed to modulate GLI transcriptional activity in cancer cells without primary cilia. However, there was also no modulation of GLI transcriptional activity in either ciliated cancer cell line. SAG treatment of SKOV3 induced localization of smoothened to primary cilia, as assessed by immunofluorescence, even though there was no increase in GLI transcriptional activity, suggesting a defect in activation of SMO in the primary cilia or in steps later in the hedgehog pathway. In contrast to SKOV3, SAG treatment of PANC1 did not cause the localization of smoothened to primary cilia. Our data demonstrate that the presence of primary cilia in the cancer epithelial cells lines tested does not indicate their responsiveness to smoothened activation or inhibition.