Hypoxia-mediated repression of pyruvate carboxylase drives immunosuppression.

BACKGROUND: Metabolic plasticity mediates breast cancer survival, growth, and immune evasion during metastasis. However, how tumor cell metabolism is influenced by and feeds back to regulate breast cancer progression are not fully understood. We identify hypoxia-mediated suppression of pyruvate carboxylase (PC), and subsequent induction of lactate production, as a metabolic regulator of immunosuppression. METHODS: We used qPCR, immunoblot, and reporter assays to characterize repression of PC in hypoxic primary tumors. Steady state metabolomics were used to identify changes in metabolite pools upon PC depletion. In vivo tumor growth and metastasis assays were used to evaluate the impact of PC manipulation and pharmacologic inhibition of lactate transporters. Immunohistochemistry, flow cytometry, and global gene expression analyzes of tumor tissue were employed to characterize the impact of PC depletion on tumor immunity. RESULTS: PC is essential for metastatic colonization of the lungs. In contrast, depletion of PC in tumor cells promotes primary tumor growth. This effect was only observed in immune competent animals, supporting the hypothesis that repression of PC can suppress anti-tumor immunity. Exploring key differences between the pulmonary and mammary environments, we demonstrate that hypoxia potently downregulated PC. In the absence of PC, tumor cells produce more lactate and undergo less oxidative phosphorylation. Inhibition of lactate metabolism was sufficient to restore T cell populations to PC-depleted mammary tumors. CONCLUSIONS: We present a dimorphic role for PC in primary mammary tumors vs. pulmonary metastases. These findings highlight a key contextual role for PC-directed lactate production as a metabolic nexus connecting hypoxia and antitumor immunity.

Pathological findings in South American camelids presented at a farm animal clinic in Northern Germany (2005-2021).

To expand the knowledge about common diseases in llamas and alpacas in Germany, a screening of the cases of South American camelids presented at the Clinic for Swine and Small Ruminants of the University of Veterinary Medicine Hannover, Germany from 2005 to the end of November 2021 was performed. A retrospective evaluation of necropsy reports from this period was conducted. Overall, necropsy reports were evaluated from 187 alpacas, 35 llamas and one vicuña (n = 223). A total of 50.2% of the dissected animals were thin or cachectic. Pathological alterations of the gastrointestinal tract were the most common findings (44.8%). In addition, liver changes were recorded, most frequently in adult animals. In contrast, diseases of the respiratory tract and the nervous system were found more frequently in juvenile animals. This study provides an overview of common pathologies in South American camelids in Germany and thus may help to recognise different disease symptoms at an early stage.

CDHu40: a novel marker gene set of neuroendocrine prostate cancer (NEPC).

Prostate cancer (PCa) is the most prevalent cancer affecting American men. Castration-resistant prostate cancer (CRPC) can emerge during hormone therapy for PCa, manifesting with elevated serum prostate-specific antigen (PSA) levels, continued disease progression, and/or metastasis to the new sites, resulting in a poor prognosis. A subset of CRPC patients shows a neuroendocrine (NE) phenotype, signifying reduced or no reliance on androgen receptor (AR) signaling and a particularly unfavorable prognosis. In this study, we incorporated computational approaches based on both gene expression profiles and protein-protein interaction (PPI) networks. We identified 500 potential marker genes, which are significantly enriched in cell cycle and neuronal processes. The top 40 candidates, collectively named as CDHu40, demonstrated superior performance in distinguishing NE prostate cancer (NEPC) and non-NEPC samples based on gene expression profiles compared to other published marker sets. Notably, some novel marker genes in CDHu40, absent in the other marker sets, have been reported to be associated with NEPC in the literature, such as DDC, FOLH1, BEX1, MAST1, and CACNA1A. Importantly, elevated CDHu40 scores derived from our predictive model showed a robust correlation with unfavorable survival outcomes in patients, indicating the potential of the CDHu40 score as a promising indicator for predicting the survival prognosis of those patients with the NE phenotype. Motif enrichment analysis on the top candidates suggests that REST and E2F6 may serve as key regulators in the NEPC progression. Significance: our study integrates gene expression variances in multiple NEPC studies and protein-protein interaction network to pinpoint a specific set of NEPC maker genes namely CDHu40. These genes and scores based on their gene expression levels effectively distinguish NEPC samples and underscore the clinical prognostic significance and potential mechanism.

Covalent Fragment Screening and Optimization Identifies the Chloroacetohydrazide Scaffold as Inhibitors for Ubiquitin C-terminal Hydrolase L1.

Dysregulation of the ubiquitin-proteasome systems is a hallmark of various disease states including neurodegenerative diseases and cancer. Ubiquitin C-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme, is expressed primarily in the central nervous system under normal physiological conditions, however, is considered an oncogene in various cancers, including melanoma, lung, breast, and lymphoma. Thus, UCHL1 inhibitors could serve as a viable treatment strategy against these aggressive cancers. Herein, we describe a covalent fragment screen that identified the chloroacetohydrazide scaffold as a covalent UCHL1 inhibitor. Subsequent optimization provided an improved fragment with single-digit micromolar potency against UCHL1 and selectivity over the closely related UCHL3. The molecule demonstrated efficacy in cellular assays of metastasis. Additionally, we report a ligand-bound crystal structure of the most potent molecule in complex with UCHL1, providing insight into the binding mode and information for future optimization.

FGFR1 Signaling Facilitates Obesity-Driven Pulmonary Outgrowth in Metastatic Breast Cancer.

Survival of dormant, disseminated breast cancer cells contributes to tumor relapse and metastasis. Women with a body mass index greater than 35 have an increased risk of developing metastatic recurrence. Herein, we investigated the effect of diet-induced obesity (DIO) on primary tumor growth and metastatic progression using both metastatic and systemically dormant mouse models of breast cancer. This approach led to increased PT growth and pulmonary metastasis. We developed a novel protocol to induce obesity in Balb/c mice by combining dietary and hormonal interventions with a thermoneutral housing strategy. In contrast to standard housing conditions, ovariectomized Balb/c mice fed a high-fat diet under thermoneutral conditions became obese over a period of 10 weeks, resulting in a 250% gain in fat mass. Obese mice injected with the D2.OR model developed macroscopic pulmonary nodules compared with the dormant phenotype of these cells in mice fed a control diet. Analysis of the serum from obese Balb/c mice revealed increased levels of FGF2 as compared with lean mice. We demonstrate that serum from obese animals, exogenous FGF stimulation, or constitutive stimulation through autocrine and paracrine FGF2 is sufficient to break dormancy and drive pulmonary outgrowth. Blockade of FGFR signaling or specific depletion of FGFR1 prevented obesity-associated outgrowth of the D2.OR model. IMPLICATIONS: Overall, this study developed a novel DIO model that allowed for demonstration of FGF2:FGFR1 signaling as a key molecular mechanism connecting obesity to breakage of systemic tumor dormancy and metastatic progression.

Ultrasonography and Infrared Thermography as a Comparative Diagnostic Tool to Clinical Examination to Determine Udder Health in Sows.

The aim of the study was to examine whether the use of infrared thermography and ultrasonography can complement or replace the clinical examination of the sows' mammary glands for pathological alterations. Sows of different parities with inconspicuous udders on palpation before and after birth (n = 35) and sows at the time of weaning (n = 107) were examined. Thermal images were taken from both sides of the udder, while ultrasound pictures were taken from four sides of the respective mammary glands. Within three weeks before birth, a statistically significant increase in the average surface temperature of the glands of about 1.54 °C and of the thickness of the parenchyma of about 1.39 cm could be observed. After weaning, in 10.3% of the examined sows, roundish hyperechogenic nodules were detected sonographically in the glands´ parenchyma. The average skin temperature above the nodules was 1.24 °C lower compared to the total skin area of the altered complex. However, scratches on the udder skin showed similar temperature changes. In two sows, the nodules remained undetected during the clinical examination. Therefore, sonography seems to be superior compared to clinical and thermographic investigations, although it proved to be very time-consuming.

Characteristics of thermal images of the mammary gland and of performance in sows differing in health status and parity.

Precision livestock farming can combine sensors and complex data to provide a simple score of meaningful productivity, pig welfare, and farm sustainability, which are the main drivers of modern pig production. Examples include using infrared thermography to monitor the temperature of sows to detect the early stages of the disease. To take account of these drivers, we assigned 697 hybrid (BHZP db. Viktoria) sows to four parity groups. In addition, by pooling clinical findings from every sow and their piglets, sows were classified into three groups for the annotation: healthy, clinically suspicious, and diseased. Besides, the udder was thermographed, and performance data were documented. Results showed that the piglets of diseased sows with eighth or higher parity had the lowest daily weight gain [healthy; 192 g ± 31.2, clinically suspicious; 191 g ± 31.3, diseased; 148 g ± 50.3 (p < 0.05)] and the highest number of stillborn piglets (healthy; 2.2 ± 2.39, clinically suspicious; 2.0 ± 1.62, diseased; 3.91 ± 4.93). Moreover, all diseased sows showed higher maximal skin temperatures by infrared thermography of the udder (p < 0.05). Thus, thermography coupled with Artificial Intelligence (AI) systems can help identify and orient the diagnosis of symptomatic animals to prompt adequate reaction at the earliest time.

New Insights into Neutrophil Extracellular Trap (NETs) Formation from Porcine Neutrophils in Response to Bacterial Infections.

Actinobacillus pleuropneumoniae (A.pp, Gram negative) and Streptococcus (S.) suis (Gram positive) can cause severe diseases in pigs. During infection, neutrophils infiltrate to counteract these pathogens with phagocytosis and/or neutrophil extracellular traps (NETs). NETs consist of a DNA-backbone spiked with antimicrobial components. The NET formation mechanisms in porcine neutrophils as a response to both of the pathogens are not entirely clear. The aim of this study was to investigate whether A.pp (serotype 2, C3656/0271/11) and S. suis (serotype 2, strain 10) induce NETs by NADPH oxidase- or CD18-dependent mechanisms and to characterize phenotypes of NETs in porcine neutrophils. Therefore, we investigated NET induction in porcine neutrophils in the presence and absence of NET inhibitors and quantified NETs after 3 h. Furthermore, NETosis and phagocytosis were investigated by transmission electron microscopy after 30 min to characterize different phenotypes. A.pp and S. suis induce NETs that are mainly ROS-dependent. A.pp induces NETs that are partially CD18-dependent. Thirty minutes after infection, both of the pathogens induced a vesicular NET formation with only slight differences. Interestingly, some neutrophils showed only NET-marker positive phagolysosomes, but no NET-marker positive vesicles. Other neutrophils showed vesicular NETs and only NET-marker negative phagolysosomes. In conclusion, both of the pathogens induce ROS-dependent NETs. Vesicular NETosis and phagocytosis occur in parallel in porcine neutrophils in response to S. suis serotype 2 and A.pp serotype 2.

1α,25-dihydroxyvitamin D reduction of MCF10A-ras cell viability in extracellular matrix detached conditions is dependent on regulation of pyruvate carboxylase.

An emerging hallmark of cancer is cellular metabolic reprogramming to adapt to varying cellular environments. Throughout the process of metastasis cancer cells gain anchorage independence which confers survival characteristics when detached from the extracellular matrix (ECM). Previous work demonstrates that the bioactive metabolite of vitamin D, 1α,25-dihydroxyvitamin D (1,25[OH]2D), suppresses cancer progression, potentially by suppressing the ability of cells to metabolically adapt to varying cellular environments such as ECM detachment. The purpose of the present study was to determine the mechanistic bases of the effects of 1,25(OH)2D on cell survival in ECM-detached conditions. Pretreatment of MCF10A-ras breast cancer cells for 3 d with 1,25(OH)2D reduced the viability of cells in subsequent detached conditions by 11%. Enrichment of 13C5-glutamine was reduced in glutamate (21%), malate (30%), and aspartate (23%) in detached compared to attached MCF10A-ras cells. Pretreatment with 1,25(OH)2D further reduced glutamine flux into downstream metabolites glutamate (5%), malate (6%), and aspartate (10%) compared to detached vehicle treated cells. Compared to attached cells, detachment increased pyruvate carboxylase (PC) mRNA abundance and protein expression by 95% and 190%, respectively. Consistent with these results, 13C6-glucose derived M+3 labelling was shown to preferentially replenish malate and aspartate, but not citrate pools, demonstrating increased PC activity in detached cells. In contrast, 1,25(OH)2D pretreatment of detached cells reduced PC mRNA abundance and protein expression by 63% and 56%, respectively, and reduced PC activity as determined by decreased 13C6-glucose derived M+3 labeling in citrate (8%) and aspartate (50%) pools, relative to vehicle-treated detached cells. While depletion of PC with doxycycline-inducible shRNA reduced detached cell viability, PC knockdown in combination with 1,25(OH)2D treatment did not additionally affect the viability of detached cells. Further, PC overexpression improved detached cell viability, and inhibited the effect of 1,25(OH)2D on detached cell survival, suggesting that 1,25(OH)2D mediates its effects in detachment through regulation of PC expression. These results suggest that inhibition of PC by 1,25(OH)2D suppresses cancer cell anchorage independence.

Hypoxia-Mediated ATF4 Induction Promotes Survival in Detached Conditions in Metastatic Murine Mammary Cancer Cells.

Regions of hypoxia are common in solid tumors and drive changes in gene expression that increase risk of cancer metastasis. Tumor cells must respond to the stress of hypoxia by activating genes to modify cell metabolism and antioxidant response to improve survival. The goal of the current study was to determine the effect of hypoxia on cell metabolism and markers of oxidative stress in metastatic (metM-Wntlung) compared with nonmetastatic (M-Wnt) murine mammary cancer cell lines. We show that hypoxia induced a greater suppression of glutamine to glutamate conversion in metastatic cells (13% in metastatic cells compared to 7% in nonmetastatic cells). We also show that hypoxia increased expression of genes involved in antioxidant response in metastatic compared to nonmetastatic cells, including glutamate cysteine ligase catalytic and modifier subunits and malic enzyme 1. Interestingly, hypoxia increased the mRNA level of the transaminase glutamic pyruvic transaminase 2 (Gpt2, 7.7-fold) only in metM-Wntlung cells. The change in Gpt2 expression was accompanied by transcriptional (4.2-fold) and translational (6.5-fold) induction of the integrated stress response effector protein activating transcription factor 4 (ATF4). Genetic depletion ATF4 demonstrated importance of this molecule for survival of hypoxic metastatic cells in detached conditions. These findings indicate that more aggressive, metastatic cancer cells utilize hypoxia for metabolic reprogramming and induction of antioxidant defense, including activation of ATF4, for survival in detached conditions.

Gastric Ulcers in Alpacas- Clinical, Laboratory, and Pathological Findings.

Gastric ulcers are a common finding in post-mortem examinations of South American camelids (SAC), but diagnosis in living animals is often difficult. The aim of this study was to provide an overview of the incidence of gastric ulcers in alpacas, common concomitant diseases, and clinical as well as laboratory findings to facilitate diagnosis for veterinarians. For this purpose, a total of 187 necropsy reports of alpacas were evaluated, including clinical and laboratory findings on the living animal. A total of 23.5% of the animals (n = 44) were found to have gastric ulcers, nine were perforated. Compartment 3 was most frequently affected by gastric ulcers. No sex predilection could be detected, but animals 1 year of age and older were more frequently affected by gastric ulcers than animals under 1 year of age. Alpacas with gastric ulcers were presented to the clinic due to different non-specific symptoms. In alpacas with gastric ulcers, significantly more organs or organ systems besides the stomach revealed clinical findings than in animals without gastric ulcers. Of the 44 animals with gastric ulcers, a total of 21 alpacas (47.7%) had a poor nutritional status, but cachexia was not significantly more frequent in animals with gastric ulcers than in other dissected animals without ulcers. Hematologic investigations revealed a significantly lower white blood count and significantly lower segmented neutrophils than in deceased animals without ulcers. Compared to animals discharged after treatment, alpacas that died with gastric ulcers had significantly higher levels of band neutrophils and fewer eosinophils and basophils. Occult blood in feces was found in three of 12 animals with gastric ulcers examined for occult blood. In summary, gastric ulcers are a common problem in SAC, which is difficult to diagnose clinically or by laboratory investigations. As these are often chronic processes involving other organ systems, regular monitoring of the animals' nutritional status and early detection of disease symptoms may help to prevent gastric ulcers.

Transglutaminase-2 mediates acquisition of neratinib resistance in metastatic breast cancer.

Acquisition of resistance to targeted therapies remains a major clinical obstacle for the HER2+ subtype of breast cancer. Using an isogeneic progression series of HER2+ breast cancer metastasis we demonstrate that metastatic cells have an increased capacity to acquire resistance to the covalent, pan-ErbB inhibitor, neratinib. RNA sequencing analyses comparing parental and metastatic cells identified upregulation of transglutaminase 2 (TG2). Genetic depletion and overexpression approaches established that TG2 is both necessary and sufficient for acquisition of neratinib resistance. Mechanistically, we describe a pathway in which TG2-mediates activation of NF-κB signaling leading to upregulation of IL-6 in metastatic cells. This autocrine expression of IL-6 functions to maintain enhanced levels of TG2 via JAK:STAT3 signaling. This drug persistence feedback loop can be interrupted through the use of the JAK1/2 inhibitor ruxolitinib. In vivo application of ruxolitinib had no effect on tumor growth under non-treated conditions, but effectively prevented acquisition of resistance, leading to tumor regression upon coadministration with neratinib. Overall, our studies reveal a mechanism in metastatic breast cancer cells that predisposes them to acquisition of resistance to ErbB-targeted therapeutics. Clinically, immediate application of ruxolitinib could prevent acquisition of resistance and improve patient responses to HER2-targeted therapies.

Increased Ammonium Toxicity in Response to Exogenous Glutamine in Metastatic Breast Cancer Cells.

Several cancers, including breast cancers, show dependence on glutamine metabolism. The purpose of the present study was to determine the mechanistic basis and impact of differential glutamine metabolism in nonmetastatic and metastatic murine mammary cancer cells. Universally labeled 13C5-glutamine metabolic tracing, qRT-PCR, measures of reductive-oxidative balance, and exogenous ammonium chloride treatment were used to assess glutamine reprogramming. Results show that 4 mM media concentration of glutamine, compared with 2 mM, reduced viability only in metastatic cells, and that this decrease in viability was accompanied by increased incorporation of glutamine-derived carbon into the tricarboxylic acid (TCA) cycle. While increased glutamine metabolism in metastatic cells occurred in tandem with a decrease in the reduced/oxidized glutathione ratio, treatment with the antioxidant molecule N-acetylcysteine did not rescue cell viability. However, the viability of metastatic cells was more sensitive to ammonium chloride treatment compared with nonmetastatic cells, suggesting a role of metabolic reprogramming in averting nitrogen cytotoxicity in nonmetastatic cells. Overall, these results demonstrate the ability of nonmetastatic cancer cells to reprogram glutamine metabolism and that this ability may be lost in metastatic cells.

Bacteriological investigation of milk samples and biopsies from the mammary gland of healthy sows.

OBJECTIVE: Milk samples from clinically healthy sows often contain a similar bacterial content as samples from mastitic sows. To verify whether contamination during sample collection is a possible reason for this or bacterial content in the mammary gland postpartum is a regular finding, the aim of the present study was to compare the suitability of milk samples and mammary gland biopsies for assessing the bacteriological status of healthy sows. MATERIAL AND METHODS: Twenty-five clinically healthy sows of different parities were selected. The mammary skin and teats were cleaned and disinfected before biopsies and milk samples were taken from the second mammary gland on the left udder side one day postpartum. Needle biopsies were performed after local anaesthesia. Samples were investigated bacteriologically for aerobic bacteria and a semi-quantitative classification of bacterial growth was carried out. Additionally, formalin-fixed paraffin-embedded biopsies from 18 of the healthy sows were stained and scored for inflammatory cells. RESULTS: A low bacterial content could be found in 96 % of milk samples and in 92 % of biopsies from the healthy sows. Both Gram-positive (mostly streptococci and staphylococci) and Gram-negative bacteria (Escherichia coli) were detected. Histopathological examination revealed mild inflammatory cell infiltration with mainly plasma cells and lymphocytes, in rare cases neutrophilic granulocytes. CONCLUSION: Biopsies of the mammary gland provided similar results with regard to bacteriological investigation compared to milk sampling although these were collected under aseptic conditions. Therefore, it is assumed that ubiquitous bacteria are regularly present in the colostrum and in the mammary gland tissue of clinically healthy sows after parturition. Histopathological findings were not entirely uneventful. CLINICAL RELEVANCE: Milk samples and biopsies from the thoroughly cleaned and disinfected mammary gland indicate a regular bacterial load of the gland postpartum; biopsies do not provide advantages with regard to hygienic conditions.

Preclinical Development of the Class-I-Selective Histone Deacetylase Inhibitor OKI-179 for the Treatment of Solid Tumors.

Histone deacetylases (HDACs) play critical roles in epigenomic regulation, and histone acetylation is dysregulated in many human cancers. Although HDAC inhibitors are active in T-cell lymphomas, poor isoform selectivity, narrow therapeutic indices, and a deficiency of reliable biomarkers may contribute to the lack of efficacy in solid tumors. In this article, we report the discovery and preclinical development of the novel, orally bioavailable, class-I-selective HDAC inhibitor, OKI-179. OKI-179 and its cell active predecessor OKI-005 are thioester prodrugs of the active metabolite OKI-006, a unique congener of the natural product HDAC inhibitor largazole. OKI-006, OKI-005, and subsequently OKI-179, were developed through a lead candidate optimization program designed to enhance physiochemical properties without eroding potency and selectivity relative to largazole. OKI-005 displays antiproliferative activity in vitro with induction of apoptosis and increased histone acetylation, consistent with target engagement. OKI-179 showed antitumor activity in preclinical cancer models with a favorable pharmacokinetic profile and on-target pharmacodynamic effects. Based on its potency, desirable class I HDAC inhibition profile, oral bioavailability, and efficacy against a broad range of solid tumors, OKI-179 is currently being evaluated in a first-in-human phase I clinical trial with plans for continued clinical development in solid tumor and hematologic malignancies.

DNA-Functionalized Ti3C2Tx MXenes for Selective and Rapid Detection of SARS-CoV-2 Nucleocapsid Gene.

Coronavirus disease 2019 (COVID-19) is an emerging human infectious disease caused by severe acute respiratory syndrome 2 (SARS-CoV-2, initially called novel coronavirus 2019-nCoV) virus. Thus, an accurate and specific diagnosis of COVID-19 is urgently needed for effective point-of-care detection and disease management. The reported promise of two-dimensional (2D) transition-metal carbides (Ti3C2Tx MXene) for biosensing owing to a very high surface area, high electrical conductivity, and hydrophilicity informed their selection for inclusion in functional electrodes for SARS-CoV-2 detection. Here, we demonstrate a new and facile functionalization strategy for Ti3C2Tx with probe DNA molecules through noncovalent adsorption, which eliminates expensive labeling steps and achieves sequence-specific recognition. The 2D Ti3C2Tx functionalized with complementary DNA probes shows a sensitive and selective detection of nucleocapsid (N) gene from SARS-CoV-2 through nucleic acid hybridization and chemoresistive transduction. The fabricated sensors are able to detect the SARS-CoV-2 N gene with sensitive and rapid response, a detection limit below 105 copies/mL in saliva, and high specificity when tested against SARS-CoV-1 and MERS. We hypothesize that the MXenes' interlayer spacing can serve as molecular sieving channels for hosting organic molecules and ions, which is a key advantage to their use in biomolecular sensing.

Tumor Cell-Autonomous SHP2 Contributes to Immune Suppression in Metastatic Breast Cancer.

SH2 containing protein tyrosine phosphatase-2 (SHP2) is recognized as a druggable oncogenic phosphatase that is expressed in both tumor cells and immune cells. How tumor cell-autonomous SHP2 contributes to an immunosuppressive tumor microenvironment (TME) and therapeutic failure of immune checkpoint blockades in metastatic breast cancer (MBC) is not fully understood. Herein, we utilized systemic SHP2 inhibition and inducible genetic depletion of SHP2 to investigate immune reprogramming during SHP2 targeting. Pharmacologic inhibition of SHP2 sensitized MBC cells growing in the lung to α-programmed death ligand 1 (α-PD-L1) antibody treatment via relieving T-cell exhaustion induced by checkpoint blockade. Tumor cell-specific depletion of SHP2 similarly reduced pulmonary metastasis and also relieved exhaustion markers on CD8+ and CD4+ cells. Both systemic SHP2 inhibition and tumor cell-autonomous SHP2 depletion reduced tumor-infiltrated CD4+ T cells and M2-polarized tumor-associated macrophages. Analysis of TCGA datasets revealed that phosphorylation of SHP2 is important for immune-cell infiltration, T-cell activation and antigen presentation. To investigate this mechanistically, we conducted in vitro T-cell killing assays, which demonstrated that pretreatment of tumor cells with FGF2 and PDGF reduced the cytotoxicity of CD8+ T cells in a SHP2-dependent manner. Both growth factor receptor signaling and three-dimensional culture conditions transcriptionally induced PD-L1 via SHP2. Finally, SHP2 inhibition reduced MAPK signaling and enhanced STAT1 signaling, preventing growth factor-mediated suppression of MHC class I. Overall, our findings support the conclusion that tumor cell-autonomous SHP2 is a key signaling node utilized by MBC cells to engage immune-suppressive mechanisms in response to diverse signaling inputs from TME. Significance: Findings present inhibition of SHP2 as a therapeutic option to limit breast cancer metastasis by promoting antitumor immunity.

High-Throughput Magnetic Actuation Platform for Evaluating the Effect of Mechanical Force on 3D Tumor Microenvironment.

Accurately replicating and analyzing cellular responses to mechanical cues is vital for exploring metastatic disease progression. However, many of the existing in vitro platforms for applying mechanical stimulation seed cells on synthetic substrates. To better recapitulate physiological conditions, a novel actuating platform is developed with the ability to apply tensile strain on cells at various amplitudes and frequencies in a high-throughput multi-well culture plate using a physiologically-relevant substrate. Suspending fibrillar fibronectin across the body of the magnetic actuator provides a matrix representative of early metastasis for 3D cell culture that is not reliant on a synthetic substrate. This platform enables the culturing and analysis of various cell types in an environment that mimics the dynamic stretching of lung tissue during normal respiration. Metabolic activity, YAP activation, and morphology of breast cancer cells are analyzed within one week of cyclic stretching or static culture. Further, matrix degradation is significantly reduced in breast cancer cell lines with metastatic potential after actuation. These new findings demonstrate a clear suppressive cellular response due to cyclic stretching that has implications for a mechanical role in the dormancy and reactivation of disseminated breast cancer cells to macrometastases.