Respiratory complex I regulates dendritic cell maturation in explant model of human tumor immune microenvironment.

BACKGROUND: Combining cytotoxic chemotherapy or novel anticancer drugs with T-cell modulators holds great promise in treating advanced cancers. However, the response varies depending on the tumor immune microenvironment (TIME). Therefore, there is a clear need for pharmacologically tractable models of the TIME to dissect its influence on mono- and combination treatment response at the individual level. METHODS: Here we establish a patient-derived explant culture (PDEC) model of breast cancer, which retains the immune contexture of the primary tumor, recapitulating cytokine profiles and CD8+T cell cytotoxic activity. RESULTS: We explored the immunomodulatory action of a synthetic lethal BCL2 inhibitor venetoclax+metformin drug combination ex vivo, discovering metformin cannot overcome the lymphocyte-depleting action of venetoclax. Instead, metformin promotes dendritic cell maturation through inhibition of mitochondrial complex I, increasing their capacity to co-stimulate CD4+T cells and thus facilitating antitumor immunity. CONCLUSIONS: Our results establish PDECs as a feasible model to identify immunomodulatory functions of anticancer drugs in the context of patient-specific TIME.

Hepsin promotes breast tumor growth signaling via the TGFß-EGFR axis.

Hepsin, a type II transmembrane serine protease, is commonly overexpressed in prostate and breast cancer. The hepsin protein is stabilized by the Ras-MAPK pathway, and, downstream, this protease regulates the degradation of extracellular matrix components and activates growth factor pathways, such as the hepatocyte growth factor (HGF) and transforming growth factor beta (TGFß) pathway. However, how exactly active hepsin promotes cell proliferation machinery to sustain tumor growth is not fully understood. Here, we show that genetic deletion of the gene encoding hepsin (Hpn) in a WAP-Myc model of aggressive MYC-driven breast cancer inhibits tumor growth in the primary syngrafted sites and the growth of disseminated tumors in the lungs. The suppression of tumor growth upon loss of hepsin was accompanied by downregulation of TGFß and EGFR signaling together with a reduction in epidermal growth factor receptor (EGFR) protein levels. We further demonstrate in 3D cultures of patient-derived breast cancer explants that both basal TGFß signaling and EGFR protein expression are inhibited by neutralizing antibodies or small-molecule inhibitors of hepsin. The study demonstrates a role for hepsin as a regulator of cell proliferation and tumor growth through TGFß and EGFR pathways, warranting consideration of hepsin as a potential indirect upstream target for therapeutic inhibition of TGFß and EGFR pathways in cancer.

A deep convolutional neural network for efficient microglia detection.

Microglial cells are a type of glial cells that make up 10-15% of all brain cells, and they play a significant role in neurodegenerative disorders and cardiovascular diseases. Despite their vital role in these diseases, developing fully automated microglia counting methods from immunohistological images is challenging. Current image analysis methods are inefficient and lack accuracy in detecting microglia due to their morphological heterogeneity. This study presents development and validation of a fully automated and efficient microglia detection method using the YOLOv3 deep learning-based algorithm. We applied this method to analyse the number of microglia in different spinal cord and brain regions of rats exposed to opioid-induced hyperalgesia/tolerance. Our numerical tests showed that the proposed method outperforms existing computational and manual methods with high accuracy, achieving 94% precision, 91% recall, and 92% F1-score. Furthermore, our tool is freely available and adds value to exploring different disease models. Our findings demonstrate the effectiveness and efficiency of our new tool in automated microglia detection, providing a valuable asset for researchers in neuroscience.

Hepsin regulates TGFß signaling via fibronectin proteolysis.

Transforming growth factor-beta (TGFß) is a multifunctional cytokine with a well-established role in mammary gland development and both oncogenic and tumor-suppressive functions. The extracellular matrix (ECM) indirectly regulates TGFß activity by acting as a storage compartment of latent-TGFß, but how TGFß is released from the ECM via proteolytic mechanisms remains largely unknown. In this study, we demonstrate that hepsin, a type II transmembrane protease overexpressed in 70% of breast tumors, promotes canonical TGFß signaling through the release of latent-TGFß from the ECM storage compartment. Mammary glands in hepsin CRISPR knockout mice showed reduced TGFß signaling and increased epithelial branching, accompanied by increased levels of fibronectin and latent-TGFß1, while overexpression of hepsin in mammary tumors increased TGFß signaling. Cell-free and cell-based experiments showed that hepsin is capable of direct proteolytic cleavage of fibronectin but not latent-TGFß and, importantly, that the ability of hepsin to activate TGFß signaling is dependent on fibronectin. Altogether, this study demonstrates a role for hepsin as a regulator of the TGFß pathway in the mammary gland via a novel mechanism involving proteolytic downmodulation of fibronectin.

miR-22 and miR-205 Drive Tumor Aggressiveness of Mucoepidermoid Carcinomas of Salivary Glands.

OBJECTIVES: To integrate mRNA and miRNA expression profiles of mucoepidermoid carcinomas (MECs) and normal salivary gland (NSGs) tissue samples and identify potential drivers. MATERIAL AND METHODS: Gene and miRNA expression arrays were performed in 35 MECs and six NSGs. RESULTS: We found 46 differentially expressed (DE) miRNAs and 3,162 DE mRNAs. Supervised hierarchical clustering analysis of the DE transcripts revealed two clusters in both miRNA and mRNA profiles, which distinguished MEC from NSG samples. The integrative miRNA-mRNA analysis revealed a network comprising 696 negatively correlated interactions (44 miRNAs and 444 mRNAs) involving cell signaling, cell cycle, and cancer-related pathways. Increased expression levels of miR-205-5p and miR-224-5p and decreased expression levels of miR-139-3p, miR-145-3p, miR-148a-3p, miR-186-5p, miR-338-3p, miR-363-3p, and miR-4324 were significantly related to worse overall survival in MEC patients. Two overexpressed miRNAs in MEC (miR-22 and miR-205) were selected for inhibition by the CRISPR-Cas9 method. Cell viability, migration, and invasion assays were performed using an intermediate grade MEC cell line. Knockout of miR-205 reduced cell viability and enhanced ZEB2 expression, while miR-22 knockout reduced cell migration and invasion and enhanced ESR1 expression. Our results indicate a distinct transcriptomic profile of MEC compared to NSG, and the integrative analysis highlighted miRNA-mRNA interactions involving cancer-related pathways, including PTEN and PI3K/AKT. CONCLUSION: The in vitro functional studies revealed that miR-22 and miR-205 deficiencies reduced the viability, migration, and invasion of the MEC cells suggesting they are potential oncogenic drivers in MEC.

Biopsy quality is essential for preoperative prognostication in oral tongue cancer.

A role for incisional biopsy in preoperative prognostication is increasingly being advocated in oral tongue squamous cell carcinomas (OTSCC). Biopsies at two locations were compared, and prognostic factors in biopsies and their corresponding resections were evaluated. A total of 138 OTSCC biopsy slides from Finland and Saudi Arabia were compared for size (horizontal and vertical) and invasive front. The Finnish cases were assessed for tumor stroma ratio (TSR) and tumor-infiltrating lymphocytes (TILs) using light microscopy and digital image analysis assessment and compared. Furthermore, TSR, TILs, and previously analyzed budding and depth of invasion (BD) score in biopsies were compared with their evaluation in the corresponding resections. Fifty-nine percent of Finnish and 42% of Saudi Arabian biopsies were ≥ 5 mm deep, while 98% of Saudi Arabian and 76% of Finnish biopsies were ≥ 5 mm wide. Assessment of invasion front was possible in 72% of Finnish in comparison with 40% of Saudi Arabian biopsies. There was 86.8% agreement between TSR and 75% agreement between TIL evaluation using light microscopy and digital assessment. Significant agreement was obtained on comparing the TSR (p = 0.04) and BD (p < 0.001) values in biopsies and resections. Biopsies of ≥ 5 mm depth from representative OTSCC areas are essential for prognostic information. Clinical pathologists are advised to assess BD score and TSR for prognostic features in such biopsies.

Effect of Sex Steroid Hormones on Tongue Cancer Cells In Vitro.

BACKGROUND: Tongue cancer is more common in men than in women. Yet the effects of sex steroid hormones on the behaviour of oral tongue squamous cell carcinoma (OTSCC) are not well known. Matrix metalloproteinase 8 (MMP8) is expressed in OTSCC and can degrade estrogen receptors (ERs). MATERIALS AND METHODS: Western blot was used to examine the levels of ERß in OTSCC cell lines (HSC-3 and SCC-25). We evaluated the effects of estradiol and dihydrotestosterone (DHT) on HSC-3 and SCC-25 cell migration, invasion and viability. The effect of estradiol on the invasion of MMP8-overexpressing (MMP8+) and empty vector HSC-3 cells was examined using 3D spheroid invasion assay. RESULTS: Both HSC-3 and SCC-25 cells expressed ERß. In scratch assay, estradiol, but not DHT, reduced the migration and invasion of HSC-3 and SCC-25 cells. MMP8+ HSC-3 cells showed weaker invasion than empty vector cells, in line with previous reports. However, MMP8 overexpression did not alter the effect of estradiol on HSC-3 cell invasion in spheroid assay. CONCLUSION: Estradiol inhibited the migration and invasion of OTSCC cells, whereas DHT had no effect. Our data suggest that MMP8 does not modulate the effect of estradiol in OTSCC cells. However, the sex difference in OTSCC incidence might partly be due to protective actions of estradiol in epithelial cell carcinogenesis.

Morphine-3-glucuronide causes antinociceptive cross-tolerance to morphine and increases spinal substance P expression.

Morphine-3-glucuronide (M3G), the main metabolite of morphine, has been implicated in the development of tolerance and of opioid-induced hyperalgesia, both limiting the analgesic use of morphine. We evaluated the acute and chronic effects of M3G and morphine as well as development of antinociceptive cross-tolerance between morphine and M3G after intrathecal administration and assessed the expression of pain-associated neurotransmitter substance P in the spinal cord. Sprague-Dawley rats received intrathecal M3G or morphine twice daily for 6 days. Nociception and tactile allodynia were measured with von Frey filaments after acute and chronic treatments. Substance P levels in the dorsal horn of the spinal cord were determined by immunohistochemistry after 4-day treatments. Acute morphine caused antinociception as expected, whereas acute M3G caused tactile allodynia, as did both chronic M3G and morphine. Chronic M3G also induced antinociceptive cross-tolerance to morphine. M3G and morphine increased substance P levels similarly in the nociceptive laminae of the spinal cord. This study shows that chronic intrathecal M3G sensitises animals to mechanical stimulation and elevates substance P levels in the nociceptive laminae of the spinal cord. Chronic M3G also induces antinociceptive cross-tolerance to morphine. Thus, chronic M3G exposure might contribute to morphine-induced tolerance and opioid-induced hyperalgesia.

The critical effects of matrices on cultured carcinoma cells: Human tumor-derived matrix promotes cell invasive properties.

The interaction between squamous cell carcinoma (SCC) cells and the tumor microenvironment (TME) plays a major role in cancer progression. Therefore, understanding the TME is essential for the development of cancer therapies. We used four (primary and metastatic) head and neck (HN) SCC cell lines and cultured them on top of or within 5 matrices (mouse sarcoma-derived Matrigel®, rat collagen, human leiomyoma-derived Myogel, human fibronectin and human fibrin). We performed several assays to study the effects of these matrices on the HNSCC behavior, such as proliferation, migration, and invasion, as well as cell morphology, and molecular gene profile. Carcinoma cells exhibited different growth patterns depending on the matrix. While fibrin enhanced the proliferation of all the cell lines, collagen did not. The effects of the matrices on cancer cell migration were cell line dependent. Carcinoma cells in Myogel-collagen invaded faster in scratch wound invasion assay. On the other hand, in the spheroid invasion assay, three out of four cell lines invaded faster in Myogel-fibrin. These matrices significantly affected hundreds of genes and a number of pathways, but the effects were cell line dependent. The matrix type played a major role in HNSCC cell phenotype. The effects of the ECMs were either constant, or cell line dependent. Based on these results, we suggest to select the most suitable matrix, which provides the closest condition to the in vivo TME, in order to get reliable results in in vitro experiments.

In vitro humanized 3D microfluidic chip for testing personalized immunotherapeutics for head and neck cancer patients.

OBJECTIVES: Immunotherapy and personalized medicine therapeutics are emerging as promising approaches in the management of head and neck squamous cell carcinoma (HNSCC). In spite of that, there is yet no assay that could predict individual response to immunotherapy. METHODS: We manufactured an in vitro 3D microfluidic chip to test the efficacy of immunotherapy. The assay was first tested using a tongue cancer cell line (HSC-3) embedded in a human tumour-derived matrix "Myogel/fibrin" and immune cells from three healthy donors. Next, the chips were used with freshly isolated cancer cells, patients' serum and immune cells. Chips were loaded with different immune checkpoint inhibitors, PD-L1 antibody and IDO 1 inhibitor. Migration of immune cells towards cancer cells and the cancer cell proliferation rate were evaluated. RESULTS: Immune cell migration towards HSC-3 cells was cancer cell density dependent. IDO 1 inhibitor induced immune cells to migrate towards cancer cells both in HSC-3 and in two HNSCC patient samples. Efficacy of PD-L1 antibody and IDO 1 inhibitor was patient dependent. CONCLUSION: We introduced the first humanized in vitro microfluidic chip assay to test immunotherapeutic drugs against HNSCC patient samples. This assay could be used to predict the efficacy of immunotherapeutic drugs for individual patients.

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay.

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

A deep convolutional neural network approach for astrocyte detection.

Astrocytes are involved in various brain pathologies including trauma, stroke, neurodegenerative disorders such as Alzheimer's and Parkinson's diseases, or chronic pain. Determining cell density in a complex tissue environment in microscopy images and elucidating the temporal characteristics of morphological and biochemical changes is essential to understand the role of astrocytes in physiological and pathological conditions. Nowadays, manual stereological cell counting or semi-automatic segmentation techniques are widely used for the quantitative analysis of microscopy images. Detecting astrocytes automatically is a highly challenging computational task, for which we currently lack efficient image analysis tools. We have developed a fast and fully automated software that assesses the number of astrocytes using Deep Convolutional Neural Networks (DCNN). The method highly outperforms state-of-the-art image analysis and machine learning methods and provides precision comparable to those of human experts. Additionally, the runtime of cell detection is significantly less than that of other three computational methods analysed, and it is faster than human observers by orders of magnitude. We applied our DCNN-based method to examine the number of astrocytes in different brain regions of rats with opioid-induced hyperalgesia/tolerance (OIH/OIT), as morphine tolerance is believed to activate glia. We have demonstrated a strong positive correlation between manual and DCNN-based quantification of astrocytes in rat brain.

Differential Spinal and Supraspinal Activation of Glia in a Rat Model of Morphine Tolerance.

Development of tolerance is a well known pharmacological characteristic of opioids and a major clinical problem. In addition to the known neuronal mechanisms of opioid tolerance, activation of glia has emerged as a potentially significant new mechanism. We studied activation of microglia and astrocytes in morphine tolerance and opioid-induced hyperalgesia in rats using immunohistochemistry, flow cytometry and RNA sequencing in spinal- and supraspinal regions. Chronic morphine treatment that induced tolerance and hyperalgesia also increased immunoreactivity of spinal microglia in the dorsal and ventral horns. Flow cytometry demonstrated that morphine treatment increased the proportion of M2-polarized spinal microglia, but failed to impact the number or the proportion of M1-polarized microglia. In the transcriptome of microglial cells isolated from the spinal cord (SC), morphine treatment increased transcripts related to cell activation and defense response. In the studied brain regions, no activation of microglia or astrocytes was detected by immunohistochemistry, except for a decrease in the number of microglial cells in the substantia nigra. In flow cytometry, morphine caused a decrease in the number of microglial cells in the medulla, but otherwise no change was detected for the count or the proportion of M1- and M2-polarized microglia in the medulla or sensory cortex. No evidence for the activation of glia in the brain was seen. Our results suggest that glial activation associated with opioid tolerance and opioid-induced hyperalgesia occurs mainly at the spinal level. The transcriptome data suggest that the microglial activation pattern after chronic morphine treatment has similarities with that of neuropathic pain.

A Novel Small Molecule GDNF Receptor RET Agonist, BT13, Promotes Neurite Growth from Sensory Neurons in Vitro and Attenuates Experimental Neuropathy in the Rat.

Neuropathic pain caused by nerve damage is a common and severe class of chronic pain. Disease-modifying clinical therapies are needed as current treatments typically provide only symptomatic relief; show varying clinical efficacy; and most have significant adverse effects. One approach is targeting either neurotrophic factors or their receptors that normalize sensory neuron function and stimulate regeneration after nerve damage. Two candidate targets are glial cell line-derived neurotrophic factor (GDNF) and artemin (ARTN), as these GDNF family ligands (GFLs) show efficacy in animal models of neuropathic pain (Boucher et al., 2000; Gardell et al., 2003; Wang et al., 2008, 2014). As these protein ligands have poor drug-like properties and are expensive to produce for clinical use, we screened 18,400 drug-like compounds to develop small molecules that act similarly to GFLs (GDNF mimetics). This screening identified BT13 as a compound that selectively targeted GFL receptor RET to activate downstream signaling cascades. BT13 was similar to NGF and ARTN in selectively promoting neurite outgrowth from the peptidergic class of adult sensory neurons in culture, but was opposite to ARTN in causing neurite elongation without affecting initiation. When administered after spinal nerve ligation in a rat model of neuropathic pain, 20 and 25 mg/kg of BT13 decreased mechanical hypersensitivity and normalized expression of sensory neuron markers in dorsal root ganglia. In control rats, BT13 had no effect on baseline mechanical or thermal sensitivity, motor coordination, or weight gain. Thus, small molecule BT13 selectively activates RET and offers opportunities for developing novel disease-modifying medications to treat neuropathic pain.

Characterization of a new low-dose 6-hydroxydopamine model of Parkinson's disease in rat.

Intrastriatal administration of 6-hydroxydopamine (6-OHDA) induces partial degeneration of the nigrostriatal pathway, mimicking the pathology of Parkinson's disease (PD). Setting up the partial lesion model can be challenging because a number of experimental settings can be altered. This study compares seven experimental settings in a single study on d-amphetamine-induced rotations, tyrosine hydroxylase (TH)-positive neurites in the striatum, dopamine transporter (DAT)-positive neurites in the striatum, and TH-positive cells in the substantia nigra pars compacta (SNpc) in rats. Moreover, we validate a new algorithm for estimating the number of TH-positive cells. We show that the behavior and immunoreactivity vary greatly depending on the injection settings, and we categorize the lesions as progressive, stable, or regressive based on d-amphetamine-induced rotations. The rotation behavior correlated with the degree of the lesion, analyzed by immunohistochemistry; the largest lesions were in the progressive group, and the smallest lesions were in the regressive group. We establish a new low-dose partial 6-OHDA lesion model in which a total of 6 µg was distributed evenly to three sites in the striatum at a 10° angle. The administration of low-dose 6-OHDA produced stable and reliable rotation behavior and induced partial loss of striatal TH-positive and DAT-positive neurites and TH-positive cells in the SNpc. This model is highly suitable for neurorestoration studies in the search for new therapies for PD, and the new algorithm increases the efficacy for estimating the number of dopamine neurons. This study can be extremely useful for laboratories setting up the partial 6-OHDA model.