A Low-Cost Modular Imaging System for Rapid, Multiplexed Immunofluorescence Detection in Clinical Tissues.

To image 4-plex immunofluorescence-stained tissue samples at a low cost with cellular level resolution and sensitivity and dynamic range required to detect lowly and highly abundant targets, here we describe a robust, inexpensive (<$9000), 3D printable portable imaging device (Tissue Imager). The Tissue Imager can immediately be deployed on benchtops for in situ protein detection in tissue samples. Applications for this device are broad, ranging from answering basic biological questions to clinical pathology, where immunofluorescence can detect a larger number of markers than the standard H&E or chromogenic immunohistochemistry (CIH) staining, while the low cost also allows usage in classrooms. After characterizing our platform's specificity and sensitivity, we demonstrate imaging of a 4-plex immunology panel in human cutaneous T-cell lymphoma (CTCL) formalin-fixed paraffin-embedded (FFPE) tissue samples. From those images, positive cells were detected using CellProfiler, a popular open-source software package, for tumor marker profiling. We achieved a performance on par with commercial epifluorescence microscopes that are >10 times more expensive than our Tissue Imager. This device enables rapid immunofluorescence detection in tissue sections at a low cost for scientists and clinicians and can provide students with a hands-on experience to understand engineering and instrumentation. We note that for using the Tissue Imager as a medical device in clinical settings, a comprehensive review and approval processes would be required.

Evidence of brain-derived neurotrophic factor in ameliorating cancer-related cognitive impairment: A systematic review of human studies.

Brain-derived neurotrophic factor (BDNF) plays an essential role in neurogenesis and neuroplasticity and may be a key protein in cancer-related cognitive impairment (CRCI). This systematic review assessed the relationship between BDNF biomarkers and neurocognitive outcomes in cancer patients and survivors. A search in PubMed, Scopus, and PsycINFO yielded 638 articles, of which 26 were eligible. Fourteen (54 %) studied BDNF protein levels while 15 (58 %) analyzed BDNF rs6265 polymorphism. Of the nine observational studies reporting BDNF plasma/serum levels, five (56 %) exhibited a positive association between BDNF and cognitive function. One study reported intra-tumoral BDNF levels that were negatively associated with memory. For rs6265, three (20 %) of 15 studies reported an association with cognitive function with inconsistent directions. Among seven neuroimaging studies, three (43 %) demonstrated an effect of BDNF on brain function and structure. These results suggest that BDNF is a potential monitoring biomarker and druggable target for CRCI.

Three cases of hepatic epithelioid hemangioendothelioma evaluated using conventional and contrast-enhanced ultrasound: Case reports.

Hepatic epithelioid hemangioendothelioma (HEHE) is a very rare vascular endothelial cell tumor, which lacks typical clinical manifestations and specificity of imaging features. Whether the background of fatty liver and the difference in Contrast enhanced ultrasound (CEUS) characteristics between large and small lesions has not been well defined. In this case reports, we described the ultrasound image features of three patients with HEHE. These three patients with HEHE have certain similar characteristics of conventional ultrasound and CEUS. CEUS imaging features include large nodules show earlier perfusion than liver parenchyma, with rim-enhancement, nonenhancing regions in the center, while small nodules show earlier perfusion than liver parenchyma, with hyperenhancement. All nodules show faster washout than hepatic parenchyma, showing heterogeneous hypoenhancement, and more washout lesions can be found in the PVP and LP. Conventional ultrasound and CEUS not only help to improve the diagnostic confidence of HEHE of rare liver tumors, but also can guide the biopsy area, making it easier to make accurate pathological diagnosis.

Spatial transcriptomics using combinatorial fluorescence spectral and lifetime encoding, imaging and analysis.

Multiplexed mRNA profiling in the spatial context provides new information enabling basic research and clinical applications. Unfortunately, existing spatial transcriptomics methods are limited due to either low multiplexing or complexity. Here, we introduce a spatialomics technology, termed Multi Omic Single-scan Assay with Integrated Combinatorial Analysis (MOSAICA), that integrates in situ labeling of mRNA and protein markers in cells or tissues with combinatorial fluorescence spectral and lifetime encoded probes, spectral and time-resolved fluorescence imaging, and machine learning-based decoding. We demonstrate MOSAICA's multiplexing scalability in detecting 10-plex targets in fixed colorectal cancer cells using combinatorial labeling of five fluorophores with facile error-detection and removal of autofluorescence. MOSAICA's analysis is strongly correlated with sequencing data (Pearson's r = 0.96) and was further benchmarked using RNAscopeTM and LGC StellarisTM. We further apply MOSAICA for multiplexed analysis of clinical melanoma Formalin-Fixed Paraffin-Embedded (FFPE) tissues. We finally demonstrate simultaneous co-detection of protein and mRNA in cancer cells.

Exosome loaded immunomodulatory biomaterials alleviate local immune response in immunocompetent diabetic mice post islet xenotransplantation.

Foreign body response (FBR) to biomaterials compromises the function of implants and leads to medical complications. Here, we report a hybrid alginate microcapsule (AlgXO) that attenuated the immune response after implantation, through releasing exosomes derived from human Umbilical Cord Mesenchymal Stem Cells (XOs). Upon release, XOs suppress the local immune microenvironment, where xenotransplantation of rat islets encapsulated in AlgXO led to >170 days euglycemia in immunocompetent mouse model of Type 1 Diabetes. In vitro analyses revealed that XOs suppressed the proliferation of CD3/CD28 activated splenocytes and CD3+ T cells. Comparing suppressive potency of XOs in purified CD3+ T cells versus splenocytes, we found XOs more profoundly suppressed T cells in the splenocytes co-culture, where a heterogenous cell population is present. XOs also suppressed CD3/CD28 activated human peripheral blood mononuclear cells (PBMCs) and reduced their cytokine secretion including IL-2, IL-6, IL-12p70, IL-22, and TNFα. We further demonstrate that XOs mechanism of action is likely mediated via myeloid cells and XOs suppress both murine and human macrophages partly by interfering with NFκB pathway. We propose that through controlled release of XOs, AlgXO provide a promising new platform that could alleviate the local immune response to implantable biomaterials.

ß-endorphin at the intersection of pain and cancer progression: Preclinical evidence.

We examined the association between endogenous opioid ß-endorphin, cancer progression and pain in a transgenic mouse model of breast cancer, with a rat C3(1) simian virus 40 large tumor antigen fusion gene (C3TAg). C3TAg mice develop ductal epithelial atypia at 8 weeks, progression to intra-epithelial neoplasia at 12 weeks, and invasive carcinoma with palpable tumors at 16 weeks. Consistent with invasive carcinoma at 4 months of age, C3TAg mice demonstrate a significant increase in hyperalgesia compared to younger C3TAg or control FVBN mice without tumors. Our data show that the growing tumor contributes to circulating ß-endorphin. As an endogenous ligand of mu opioid receptor, ß-endorphin has analgesic activity. Paradoxically, we observed an increase in pain in transgenic breast cancer mice with significantly high circulating and tumor-associated ß-endorphin. Increased circulating ß-endorphin correlates with increasing tumor burden. ß-endorphin induced the activation of mitogenic and survival-promoting signaling pathways, MAPK/ERK 1/2, STAT3 and Akt, observed by us in human MDA-MB-231 cells suggesting a role for ß-endorphin in breast cancer progression and associated pain.

Cardiac Troponin I R193H Mutation Is Associated with Mitochondrial Damage in Cardiomyocytes.

Malfunction of myocardial mitochondria plays a crucial role in the development of cardiovascular disorders, especially hypertrophic and dilated cardiomyopathies. Cardiac troponin I (cTnI) is an important structural protein and essential to contraction and relaxation of cardiomyocytes. Recent studies suggest that mutated cTnIR193H could function as a regulatory molecule for other cell functions. This study was to determine whether mutated cTnI could contribute to mitochondrial dysfunction of cardiomyocytes. Primary cardiomyocytes were transfected with cTnIR193H adenovirus with empty vector as control. Mitochondrial structure and function were evaluated in the cells 72 h after transfection. Transmission electron microscopy examination showed mitochondria in the cardiomyocytes with R193H mutation displayed broken cristae, vacuolation, and mitophagy. Mitochondrial function studies revealed a significant decrease in complex I activity, ATP and reactive oxygen species levels, and oxygen consumption rate compared with controls. Western blot analysis demonstrated that expressions of mitochondria-related genes, including ND5 (ubiquinone oxidoreductase chain 5), LRPPRC (a leucine-rich protein of pentatricopeptide repeat family), and PGC-1α (PPARG co-activator 1 alpha), were significantly downregulated in R193H mutation cardiomyocytes compared with the control. Swelling and broken cristae were observed in the mitochondria of cardiomyocytes from cTnIR193H mutation transgenic mice with decreased mitochondrial function, not from the littermate control mice. The data from the present study demonstrated that mitochondrial structure and function were significantly impaired in cardiomyocytes with cTnIR193H mutation, suggesting that cTnI might be critically involved in maintaining the structural and functional integrity of myocardial mitochondria.

Preclinical Evaluation of a Single Intravenous Infusion of hUC-MSC (BX-U001) in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an inflammatory disease of the joints, which causes severe pain and excessive systemic circulation of harmful inflammatory cytokines. Current treatments are limited, with some patients not responding well, and some experiencing severe and detrimental side effects. Mesenchymal stem cells (MSC) are cell-based therapeutics being evaluated as potent immunomodulators in RA and may provide relief to patients not responding well to drug-based treatments. We evaluated the safety and efficacy of BX-U001 human umbilical cord tissue-derived mesenchymal stem cells (hUC-MSC) to treat RA, in support of a successful investigational new drug application. A collagen-induced arthritis (CIA) mouse model of RA was established in DBA/1 J mice. Mice from the treatment assessment group were given a tail vein infusion of hUC-MSC 24 days after primary RA induction, while control assessment (CA) group mice were given cell-free carrier solution. All animals were evaluated daily for RA symptoms via clinical scoring, blood was taken periodically for cytokine analysis, and mice were dissected at end point for histological analysis. A linear mixed model was used to compare the rate of change among groups. The clinical scores of TA group were significantly reduced compared with CA group (P < 0.01), indicating therapeutic effects. The histological scores of the joints in TA group were significantly lower than those in the CA group (P < 0.05), but had no significant difference compared with Healthy groups (P > 0.05). The concentration of (interleukin) IL-6 in TA group was significantly reduced by 80.0% (P < 0.0001) 2 days after treatment and by 93.4% at the experimental endpoint compared with levels prior to hUC-MSC injection. A single intravenous infusion of hUC-MSC (2 × 106 cells/mouse), to CIA-induced DBA/1 J mice, resulted in significant alleviation of RA symptoms and may provide significant therapeutic benefits in humans.

Preferences of Type 1 Diabetic Patients on Devices for Islet Transplantation.

Transplantation of pancreatic islets within a biomaterial device is currently under investigation in clinical trials for the treatment of patients with type 1 diabetes (T1D). Patients' preferences on such implants could guide the designs of next-generation implantable devices; however, such information is not currently available. We surveyed the preferences of 482 patients with T1D on the size, shape, visibility, and transplantation site of islet containing implants. More than 83% of participants were willing to receive autologous stem cells, and there was no significant association between implant fabricated by one's own stem cell with gender (χ2 (1, n = 468) = 0.28; P = 0.6) or with age (χ2 (4, n = 468) = 2.92; P = 0.6). Preferred location for islet transplantation within devices was under the skin (52.7%). 48.3% preferred microscopic disks, and 32.3% preferred a thin device (like a credit card). Moreover, 58.4% preferred the implant to be as small as possible, 25.4% did not care about visibility, and 16.2% preferred their implants not to be visible. Among female participants, 81% cared about the implant visibility, whereas this number was 64% for male respondents (χ2 test (1, n = 468) = 16.34; P < 0.0001). 22% of those younger than 50 years of age and 30% of those older than 50 did not care about the visibility of implant (χ2 test (4, n = 468) = 23.69; P < 0.0001). These results suggest that subcutaneous sites and micron-sized devices are preferred choices among patients with T1D who participated in our survey.

Correlation Analysis of Conventional Ultrasound Characteristics and Strain Elastography with Ki-67 Status in Breast Cancer.

Our objectives were to measure the relationships between conventional ultrasound features, strain elastography in breast cancer and Ki-67 index and to identify parameters that predict Ki-67 index. We included 181 lesions of 178 patients who underwent surgery for breast cancer at Xianyang Central Hospital. In multivariate logistic regression analysis, strain elastography and axillary-node metastasis showed significant Ki-67 index values; the overall theoretical prediction percentage correct was 75.7%. Strain elastography showed that the median Ki-67 index in the hard group was higher than that in the soft group, and the Ki-67 index increased with increasing elasticity score. This finding may guide ultrasound-guided breast tumor biopsy for selection of puncture regions. The combined use of the Ki-67 index for strain-elastography prediction and puncture-biopsy pathology reports may increase the accuracy of clinical treatment.

CAR-T design: Elements and their synergistic function.

Chimeric antigen receptor (CAR) T cells use re-engineered cell surface receptors to specifically bind to and lyse oncogenic cells. Two clinically approved CAR-T-cell therapies have significant clinical efficacy in treating CD19-positive B cell cancers. With widespread interest to deploy this immunotherapy to other cancers, there has been great research activity to design new CAR structures to increase the range of targeted cancers and anti-tumor efficacy. However, several obstacles must be addressed before CAR-T-cell therapies can be more widely deployed. These include limiting the frequency of lethal cytokine storms, enhancing T-cell persistence and signaling, and improving target antigen specificity. We provide a comprehensive review of recent research on CAR design and systematically evaluate design aspects of the four major modules of CAR structure: the ligand-binding, spacer, transmembrane, and cytoplasmic domains, elucidating design strategies and principles to guide future immunotherapeutic discovery.

Optimization of a syngeneic murine model of bone metastasis.

Many cancers metastasize to the bones, particularly in cases of breast and prostate cancers. Due to the "vicious cycle" of cancer cells inducing bone resorption, which promotes further tumor growth, they are difficult to treat and may lead to extreme pain. These factors increase the urgency for emerging therapeutics that target bone metastases more specifically and effectively. Animal studies are essential to the development of any therapeutics, but also require robust animal models of human diseases. Robust animal models are often challenging to develop in the case of bone metastasis studies. Previous methods to induce bone metastasis include intracardiac, intravenous, subcutaneous via mammary fat pad, and intraosseous cancer cell injections, but these methods all have limitations. By contrast, the caudal artery route of injection offers more robust bone metastasis, while also resulting in a lower rate of vital organ metastases than that of other routes of tumor implantation. A syngeneic animal model of bone metastasis is necessary in many cancer studies, because it allows the use of immunocompetent animals, which more accurately mimic cancer development observed in immunocompetent humans. Here we present a detailed method to generate robust and easily monitored 4T1-CLL1 syngeneic bone metastases with over 95% occurrence in BALB/c mice, within two weeks. This method can potentially increase consistency between animals in bone cancer metastasis studies and reduce the number of animals needed for studying bone metastases in mice.

Rapid isolation of rare targets from large fluid volumes.

Rapidly isolating rare targets from larger, clinically relevant fluid volumes remains an unresolved problem in biomedicine and diagnosis. Here, we describe how 3D particle sorting can enrich targets at ultralow concentrations over 100-fold within minutes not possible with conventional approaches. Current clinical devices based on biochemical extraction and microfluidic solutions typically require high concentrations and/or can only process sub-milliliter volumes in time. In a proof-of-concept application, we isolated bacteria from whole blood as demanded for rapid sepsis diagnosis where minimal numbers of bacteria need to be found in a 1-10 mL blood sample. After sample encapsulation in droplets and target enrichment with the 3D particle sorter within a few minutes, downstream analyses were able to identify bacteria and test for antibiotic susceptibility, information which is critical for successful treatment of bloodstream infections.

Controlled Release of Stem Cell Secretome Attenuates Inflammatory Response against Implanted Biomaterials.

Inflammatory response against implanted biomaterials impairs their functional integration and induces medical complications in the host's body. To suppress such immune responses, one approach is the administration of multiple drugs to halt inflammatory pathways. This challenges patient's adherence and can cause additional complications such as infection. Alternatively, biologics that regulate multiple inflammatory pathways are attractive agents in addressing the implants immune complications. Secretome of mesenchymal stromal cells (MSCs) is a multipotent biologic, regulating the homeostasis of lymphocytes and leukocytes. Here, it is reported that alginate microcapsules loaded with processed conditioned media (pCM-Alg) reduces the infiltration and/or expression of CD68+ macrophages likely through the controlled release of pCM. In vitro cultures revealed that alginate can dose dependently induce macrophages to secrete TNFα, IL-6, IL-1ß, and GM-CSF. Addition of pCM to the cultures attenuates the secretion of TNFα (p = 0.023) and IL-6 (p < 0.0001) by alginate or lipopolysaccharide (LPS) stimulations. Mechanistically, pCM suppressed the NfκB pathway activation of macrophages in response to LPS (p < 0.0001) in vitro and cathepsin activity (p = 0.005) in response to alginate in vivo. These observations suggest the efficacy of using MSC-derived secretome to prevent or delay the host rejection of implants.

Isolation and characterization of microvesicles from mesenchymal stem cells.

Mesenchymal stem or stromal cells are currently under clinical investigation for multiple diseases. While their mechanism of action is still not fully elucidated, vesicles secreted by MSCs are believed to recapitulate their therapeutic potentials to some extent. Microvesicles (MVs), also called as microparticles or ectosome, are among secreted vesicles that could transfer cytoplasmic cargo, including RNA and proteins, from emitting (source) cells to recipient cells. Given the importance of MVs, we here attempted to establish a method to isolate and characterize MVs secreted from unmodified human bone marrow derived MSCs (referred to as native MSCs, and their microvesicles as Native-MVs) and IFNγ stimulated MSCs (referred to as IFNγ-MSCs, and their microvesicles as IFNγ-MVs). We first describe an ultracentrifugation technique to isolate MVs from the conditioned cell culture media of MSCs. Next, we describe characterization and quality control steps to analyze the protein and RNA content of MVs. Finally, we examined the potential of MVs to exert immunomodulatory effects through induction of regulatory T cells (Tregs). Secretory vesicles from MSCs are promising alternatives for cell therapy with applications in drug delivery, regenerative medicine, and immunotherapy.

Meta-analysis of preclinical studies of mesenchymal stromal cells to treat rheumatoid arthritis.

BACKGROUND: This study aims to evaluate the quality of preclinical data, determine the effect sizes, and identify experimental measures that inform efficacy using mesenchymal stromal (or stem) cells (MSC) therapy in animal models of rheumatoid arthritis (RA). METHODS: Literature searches were performed on MSC preclinical studies to treat RA. MSC treatment effect sizes were determined by the most commonly used outcome measures, including paw thickness, clinical score, and histological score. FINDINGS: A total of 48 studies and 94 treatment arms were included, among which 42 studies and 79 treatment arms reported that MSC improved outcomes. The effect sizes of RA treatments using MSC, when compared to the controls, were: paw thickness was ameliorated by 53.6% (95% confidence interval (CI): 26.7% -80.4%), histological score was decreased by 44.9% (95% CI: 33.3% -56.6%), and clinical score was decreased by 29.9% (95% CI: 16.7% -43.0%). Specifically, our results indicated that human umbilical cord derived MSC led to large improvements of the clinical score (-42.1%) and histological score (-51.4%). INTERPRETATION: To the best of our knowledge, this meta-analysis is to quantitatively answer whether MSC represent a robust RA treatment in animal models. It suggests that in preclinical studies, MSC have consistently exhibited therapeutic benefits. The findings demonstrate a need for considering variations in different animal models and treatment protocols in future studies using MSC to treat RA in humans to maximise the therapeutic gains in the era of precision medicine. FUNDS: NIH [1DP2CA195763], Baylx Inc.: BI-206512, NINDS/NIH Training Grant [Award# NS082174].

Combinatorial targeting of cancer bone metastasis using mRNA engineered stem cells.

BACKGROUND: Bone metastases are common and devastating to cancer patients. Existing treatments do not specifically target the disease sites and are therefore ineffective and systemically toxic. Here we present a new strategy to treat bone metastasis by targeting both the cancer cells ("the seed"), and their surrounding niche ("the soil"), using stem cells engineered to home to the bone metastatic niche and to maximise local delivery of multiple therapeutic factors. METHODS: We used mesenchymal stem cells engineered using mRNA to simultaneously express P-selectin glycoprotein ligand-1 (PSGL-1)/Sialyl-Lewis X (SLEX) (homing factors), and modified versions of cytosine deaminase (CD) and osteoprotegerin (OPG) (therapeutic factors) to target and treat breast cancer bone metastases in two mouse models, a xenograft intratibial model and a syngeneic model of spontaneous bone metastasis. FINDINGS: We first confirmed that MSC engineered using mRNA produced functional proteins (PSGL-1/SLEX, CD and OPG) using various in vitro assays. We then demonstrated that mRNA-engineered MSC exhibit enhanced homing to the bone metastatic niche likely through interactions between PSGL-1/SLEX and P-selectin expressed on tumour vasculature. In both the xenograft intratibial model and syngeneic model of spontaneous bone metastasis, engineered MSC can effectively kill tumour cells and preserve bone integrity. The engineered MSC also exhibited minimal toxicity in vivo, compared to its non-targeted chemotherapy counterpart (5-fluorouracil). INTERPRETATION: Our combinatorial targeting of both the cancer cells and the niche represents a simple, safe and effective way to treat metastatic bone diseases, otherwise difficult to manage with existing strategies. It can also be applied to other cell types (e.g., T cells) and cargos (e.g., genome editing components) to treat a broad range of cancer and other complex diseases. FUND: National Institutes of Health, National Cancer Institute of the National Institutes of Health, Department of Defense, California Institute of Regenerative Medicine, National Science Foundation, Baylx Inc., and Fondation ARC pour la recherche sur le cancer.

Transplantation of stem cells from umbilical cord blood as therapy for type I diabetes.

In recent years, human umbilical cord blood has emerged as a rich source of stem, stromal and immune cells for cell-based therapy. Among the stem cells from umbilical cord blood, CD45+ multipotent stem cells and CD90+ mesenchymal stem cells have the potential to treat type I diabetes mellitus (T1DM), to correct autoimmune dysfunction and replenish ß-cell numbers and function. In this review, we compare the general characteristics of umbilical cord blood-derived multipotent stem cells (UCB-SCs) and umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) and introduce their applications in T1DM. Although there are some differences in surface marker expression between UCB-SCs and UCB-MSCs, the two cell types display similar functions such as suppressing function of stimulated lymphocytes and imparting differentiation potential to insulin-producing cells (IPCs) in the setting of low immunogenicity, thereby providing a promising and safe approach for T1DM therapy.

Stem Cell-Derived Exosomes as Nanotherapeutics for Autoimmune and Neurodegenerative Disorders.

To dissect therapeutic mechanisms of transplanted stem cells and develop exosome-based nanotherapeutics in treating autoimmune and neurodegenerative diseases, we assessed the effect of exosomes secreted from human mesenchymal stem cells (MSCs) in treating multiple sclerosis using an experimental autoimmune encephalomyelitis (EAE) mouse model. We found that intravenous administration of exosomes produced by MSCs stimulated by IFNγ (IFNγ-Exo) (i) reduced the mean clinical score of EAE mice compared to PBS control, (ii) reduced demyelination, (iii) decreased neuroinflammation, and (iv) upregulated the number of CD4+CD25+FOXP3+ regulatory T cells (Tregs) within the spinal cords of EAE mice. Co-culture of IFNγ-Exo with activated peripheral blood mononuclear cells (PBMCs) cells in vitro reduced PBMC proliferation and levels of pro-inflammatory Th1 and Th17 cytokines including IL-6, IL-12p70, IL-17AF, and IL-22 yet increased levels of immunosuppressive cytokine indoleamine 2,3-dioxygenase. IFNγ-Exo could also induce Tregs in vitro in a murine splenocyte culture, likely mediated by a third-party accessory cell type. Further, IFNγ-Exo characterization by deep RNA sequencing suggested that IFNγ-Exo contains anti-inflammatory RNAs, where their inactivation partially hindered the exosomes potential to induce Tregs. Furthermore, we found that IFNγ-Exo harbors multiple anti-inflammatory and neuroprotective proteins. These results not only shed light on stem cell therapeutic mechanisms but also provide evidence that MSC-derived exosomes can potentially serve as cell-free therapies in creating a tolerogenic immune response to treat autoimmune and central nervous system disorders.