BackgroundInfectious agents are causing 20%–30% of all cancers.1 Non-human genes can act as effective tumor-specific targets.2–5 For an oncovirus, such as the Epstein-Barr virus (EBV), the viral ...genome is integrated into the cell genome and express viral proteins during the latency phase, which could be targets for immune therapies and other types of therapy. For example, therapies using autologous T lymphocytes targeting the EBV latent membrane proteins LMP1 and LMP2 have had impressive results in some lymphomas.2 MethodsStandard RNA-sequencing pipelines map the reads only to the human genome and do not, therefore, identify tumor-specific non-human antigens. Furthermore, the findings lack clinical validation.We demonstrate that our clinically validated OneRNA® platform, which leverages RNA-sequencing in tissue and blood, can identify unique viruses and their expressed mRNAs, some of which could act as tumor-specific antigens. The OneRNA® platform is validated according to CLIA standards.In this work, we identify and quantify viral RNA expressed in solid as well as in hematological tumors using an augmented version of the OneRNA® bioinformatics pipeline. We further show how we clinically validated these findings, enabling large-scale implementation in the clinic.ResultsOneRNA® in FFPE demonstrated higher sensitivity to most viruses discovered than Truseq perform on FF samples from the same tumors. Truseq is not recommended in FFPE thus OneRNA® provides a clinically validated chemistry that can effectively detect viral and possibly other infectious agents in FFPE tumor tissue. Virus was detected in all of the samples, tumor and normal tissue.Cytomegalovirus (CMV) reads were found in 70% of the breast cancer samples. However, only two viruses were found in significantly higher normalized read counts than normal tissue while CMV normalized read counts were similar to normal tissue.ConclusionsThe identification and validation of the presence of additional vaccine targets for e.g. mRNA vaccines provides an exciting opportunity for biotech and pharma to improve the response rate of checkpoint inhibitors as recently seen with Moderna’s mRNA vaccine in melanoma. It also provides the opportunity to move immune therapy and the use of check-point inhibitors into low mutation frequency (low-mut) tumors such as breast cancer by providing the immune system with a non-human target as Neo-antigens are low in low-mut tumors. Finally, it can provide the opportunity to treat the tumor if the infectious agent is treatable with existing drugs.Referenceszur Hausen H. The search for infectious causes of human cancers: Where and why. Virology, 2009;392(1):1–10. https://doi.org/10.1016/j.virol.2009.06.001Bollard CM, Gottschalk S, Torrano V, Diouf O, Ku S, Hazrat Y, Carrum G, Ramos C, Fayad L, Shpall EJ, Pro B, Liu H, Wu MF, Lee D, Sheehan AM, Zu Y, Gee AP, Brenner MK, Heslop HE, Rooney CM. Sustained complete responses in patients with lymphoma receiving autologous cytotoxic T lymphocytes targeting Epstein-Barr virus latent membrane proteins. Journal of clinical oncology: official journal of the American Society of Clinical Oncology, 2014;32(8):798–808. https://doi.org/10.1200/JCO.2013.51.5304Narunsky-Haziza L, Sepich-Poore GD, Livyatan I, Asraf O, Martino C, Nejman D, Gavert N, Stajich JE, Amit G, González A, Wandro S, Perry G, Ariel R, Meltser A, Shaffer JP, Zhu Q, Balint-Lahat N, Barshack I, Dadiani M, Gal-Yam EN, … Straussman R. Pan-cancer analyses reveal cancer-type-specific fungal ecologies and bacteriome interactions. Cell, 2022;185(20):3789–3806.e17. https://doi.org/10.1016/j.cell.2022.09.005Torres HA, Economides MP, Angelidakis G, Hosry J, Kyvernitakis A, Mahale P, Jiang Y, Miller E, Blechacz B, Naing A, Samaniego F, Kaseb A, Raad II, Granwehr BP. Sofosbuvir-based therapy in hepatitis C virus-infected cancer patients: A prospective observational study. American Journal of Gastroenterology, 2018;114(2);250–257. https://doi.org/10.1038/s41395-018-0383-2Anshuman Panda et al, Immune Activation and Benefit From Avelumab in EBV-Positive Gastric Cancer, JNCI: Journal of the National Cancer Institute, 2018;110(3):316–320, https://doi.org/10.1093/jnci/djx213Ethics ApprovalSamples was obtained from a Biobank with all the required approvals
BackgroundNon-Hodgkin lymphoma (NHL) is one of the most common cancers accounting for about 4% of all cancers. The 10-year survival rate has dramatically increased to 90% due to novel drugs including ...Rituximab targeting unique B-cell surface markers such as CD20 and CAR-T cells targeting CD19, however, most of these drugs are not differentiating between normal B-cells and malignant B-cells leaving the patient with low B-cell count, and susceptibility for infections. Most patients relapse, and the disease remains incurable.MethodsWe developed novel protocols for the enrichment and preservation of live B-cells and T-cells from blood. The live B-cells were tested in a live cell assay with drugs approved in FL. The same B-cells as well as collected lymph node biopsies were analyzed using our clinically validated OneRNA® platform to identify new targets, and biomarkers for response and repurpose drugs approved outside FL which could then be tested in the same live cell assay. We further analyzed whole blood, B-cells, and T-cells as well as biopsies using several MRD assays longitudinally during several treatment cycles and combined the data sets.ResultsWe were able to identify and rank approved treatments in FL with the results of the live cell assay as well as the OneRNA® data. The data correlated with the response to treatment. The OneRNA® assay was able to identify additional approved treatment options that were then tested in the live cell assay alone and in combination with drugs approved in FL. We further demonstrated our MRD assay was able to detect disease earlier than standard-of-care molecular assays and PET scans.ConclusionsAlthough Follicular Lymphoma is treatable, the optimal treatment needs to be individualized based on more comprehensive biomarkers assays guiding treatment options together with MRD assays detecting response and relapse.Hematological cancers provide the opportunity to enrich for malignant cells and test using more comprehensive biomarker assays such as OneRNA® that can guide response to treatment and test those treatments and treatment combinations on the patient’s own cells prior to treatment.We also validated an assay for Minimal Residual Disease (MRD), which deserves further studies as a tool to refine the clinical/metabolic response and to modulate treatment intensity/durationFinally, the OneRNA® assay provides an opportunity to identify novel targets and surface proteins that could be developed into drugs that are specifically targeting the malignant B-cells.Ethics ApprovalWe obtained samples from biobanksConsentWe obtained samples from biobanks
Key points
Exogenous Na+/H+ exchanger 1 (NHE1) expression stimulated the collective migration of epithelial cell sheets
Stimulation with epidermal growth factor, a key morphogen, primarily increased ...migration of the front row of cells, whereas NHE1 increased that of submarginal cell rows, and the two stimuli were additive
Accordingly, NHE1 localized not only to the leading edges of leader cells, but also in cryptic lamellipodia in submarginal cell rows
NHE1 expression disrupted the morphology of epithelial cell sheets and three‐dimensional cysts
Collective cell migration plays essential roles in embryonic development, in normal epithelial repair processes, and in many diseases including cancer. The Na+/H+ exchanger 1 (NHE1, SLC9A1) is an important regulator of motility in many cells and has been widely studied for its roles in cancer, although its possible role in collective migration of normal epithelial cells has remained unresolved. In the present study, we show that NHE1 expression in MDCK‐II kidney epithelial cells accelerated collective cell migration. NHE1 localized to the leading edges of leader cells, as well as to cryptic lamellipodia in submarginal cell rows. Epidermal growth factor, a kidney morphogen, increased displacement of the front row of collectively migrating cells and reduced the number of migration fingers. NHE1 expression increased the number of migration fingers and increased displacement of submarginal cell rows, resulting in additive effects of NHE1 and epidermal growth factor. Finally, NHE1 expression resulted in disorganized development of MDCK‐II cell cysts. Thus, NHE1 contributes to collective migration and epithelial morphogenesis, suggesting roles for the transporter in embryonic and early postnatal development.
Key points
Exogenous Na+/H+ exchanger 1 (NHE1) expression stimulated the collective migration of epithelial cell sheets
Stimulation with epidermal growth factor, a key morphogen, primarily increased migration of the front row of cells, whereas NHE1 increased that of submarginal cell rows, and the two stimuli were additive
Accordingly, NHE1 localized not only to the leading edges of leader cells, but also in cryptic lamellipodia in submarginal cell rows
NHE1 expression disrupted the morphology of epithelial cell sheets and three‐dimensional cysts
Display omitted
In cutaneous drug delivery, it is widely accepted that the choice of excipients affects the delivery of a drug molecule to the skin. MALDI mass spectrometry imaging (MALDI-MSI) is an ...imaging technique which enables the simultaneous detection of multiple compounds. MALDI-MSI was applied to study the penetration of tofacitinib and excipients in porcine skin from two formulations with sodium lauryl sulphate (SLS) and dexpanthenol (DXP) using Franz diffusion cells. Further, the receptor media was collected for analysis of the permeated amounts of tofacitinib and excipients.
The MALDI images showed DXP to be co-localized with tofacitinib in the epidermal and deep dermal region while SLS was distributed in the entire skin compartment. The permeation of tofacitinib for the two formulations was similar after 24 h, whereas, the percentage of permeated DXP was higher than for SLS.
This study provided an overview of the skin penetration and permeation of drug molecule and excipients. MALDI-MSI showed differences in the DXP and SLS distribution. This indicates that the excipients interact with the skin through different mechanisms. Compound-specific imaging methods such as MALDI-MSI are potential tools to increase the understanding of the complex interplay between skin, excipients and the drug molecule for optimized cutaneous drug delivery.
Foodborne Enteropathogenic Escherichia coli (EPEC) infections of the small intestine cause diarrhea especially in children and are a major cause of childhood death in developing countries. EPEC ...infects the apical membrane of the epithelium of the small intestine by attaching, effacing the microvilli under the bacteria and then forming microcolonies on the cell surface. We first asked the question where on epithelial cells EPEC attaches and grows. Using models of polarized epithelial monolayers, we evaluated the sites of initial EPEC attachment to the apical membrane and found that EPEC preferentially attached over the cell-cell junctions and formed microcolonies preferentially where three cells come together at tricellular tight junctions. The ability of EPEC to adhere increased when host cell polarity was compromised yielding EPEC access to basolateral proteins. EPEC pedestals contain basolateral cytoskeletal proteins. Thus, we asked if attached EPEC causes reorganization the protein composition of the host cell plasma membrane at sites of microcolony formation. We found that EPEC microcolony growth at the apical membrane resulted in a local accumulation of basolateral plasma membrane proteins surrounding the microcolony. Basolateral marker protein aquaporin-3 localized to forming EPEC microcolonies. Components of the basolateral vesicle targeting machinery were re-routed. The Exocyst (Exo70) was recruited to individual EPEC as was the basolateral vesicle SNARE VAMP-3. Moreover, several Rab variants were also recruited to the infection site, and their dominant-negative equivalents were not. To quantitatively study the recruitment of basolateral proteins, we created a pulse of the temperature sensitive basolateral VSVG, VSVG3-SP-GFP, from the trans-Golgi Network. We found that after release from the TGN, significantly more VSVG3-SP-GFP accumulated at the site of microcolony growth than on equivalent membrane regions of uninfected cells. This suggests that trafficking of vesicles destined for the basolateral membrane are redirected to the apical site of microcolony growth. Thus, in addition to disrupting host cell fence function, local host cell plasma membrane protein composition is changed by altered protein trafficking and recruitment of basolateral proteins to the apical microcolony. This may aid EPEC attachment and subsequent microcolony growth.
We determined the impact of three factors on mortality in HIV-infected patients who had been on highly active antiretroviral therapy (HAART) for at least one year: (1) insufficient response to ...(HAART) and presence of AIDS-defining diseases, (2) comorbidity, and (3) drug and alcohol abuse and compared the mortality to that of the general population.
In a Danish nationwide, population-based cohort study, we used population based registries to identify (1) all Danish HIV-infected patients who started HAART in the period 1 January 1998-1 July 2009, and (2) a comparison cohort of individuals matched on date of birth and gender (N = 2,267 and 9,068, respectively). Study inclusion began 1 year after start of HAART. Patients were categorised hierarchically in four groups according to the three risk factors, which were identified before study inclusion. The main outcome measure was probability of survival from age 25 to 65 years. The probability of survival from age 25 to age 65 was substantially lower in HIV patients 0.48 (95% confidence interval (CI) 0.42-0.55) compared to the comparison cohort 0.88 (0.86 to 0.90). However, in HIV patients with no risk factors (N = 871) the probability of survival was equivalent to that of the general population 0.86 (95% CI 0.77-0.92). In contrast, the probability of survival was 0.58 in patients with HIV risk factors (N = 704), 0.30 in patients with comorbidities (N = 479), and 0.03 in patients with drug or alcohol abuse (N = 313).
The increased risk of death in HIV-infected individuals is mainly attributable to risk factors that can be identified prior to or in the initial period of antiretroviral treatment. Mortality in patients without risk factors on a successful HAART is almost identical to that of the non-HIV-infected population.
Display omitted
In skin penetration studies, HPLC-MS/MS analysis on extracts of heat-separated epidermis and dermis provides an estimate of the amount of drug penetrated. In this study, MALDI-MSI ...enabled qualitative skin distribution analysis of endogenous molecules and the drug molecule, tofacitinib and quantitative analysis of the amount of tofacitinib in the epidermis. The delivery of tofacitinib to the skin was investigated in a Franz diffusion cell using three different formulations (two oil-in-water creams, C1 and C2 and an aqueous gel). Further, in vitro release testing (IVRT) was performed and resulted in the fastest release of tofacitinib from the aqueous gel and the lowest from C2. In the ex vivo skin penetration and permeation study, C1 showed the largest skin retention of tofacitinib, whereas, lower retention and higher permeation were observed for the gel and C2. The quantitative MALDI-MSI analysis showed that the content of tofacitinib in the epidermis for the C1 treated samples was comparable to HPLC-MS/MS analysis, whereas, the samples treated with C2 and the aqueous gel were below LOQ. The study demonstrates that MALDI-MSI can be used for the quantitative determination of drug penetration in epidermis, as well as, to provide valuable information on qualitative skin distribution of tofacitinib.
ABSTRACT
Plasticity of epithelial cell‐cell adhesion is vital in epithelial homeostasis and is regulated in multiple processes associated with cell migration, such as embryogenesis and wound healing. ...In cancer, cell‐cell adhesion is compromised and is associated with increased cell migration and metastasis. Aquaporin (AQP) water channels facilitate water transport across cell membranes and are essential in the regulation of body water homeostasis. Increased expression of several AQPs, especially AQP5, is associated with increased cancer cell migration, metastasis, and poor prognosis. We found that AQP5 overexpression in normal epithelial cells induced cell detachment and dissemination from migrating cell sheets. AQP5 reduced both cell‐cell coordination during collective migration and overall distance covered by the migrating cell sheets. AQP5 and the isoforms AQP1 and AQP4 decreased, whereas AQP3 increased, levels of plasma membrane‐associated lateral junctional proteins. This regulation was mediated by the cytoplasmic domains of the AQPs. This shows that the AQPs have dual functions in epithelial physiology: as channel proteins and as differential regulators of cell‐cell adhesiveness. This regulation may contribute to dynamic regulation of cell junctions in processes such as embryogenesis and wound healing and also explain the pivotal roles of AQPs in carcinogenesis and metastasis.—Login, F. H., Jensen, H. H., Pedersen, G. A., Koffman, J. S., Kwon, T.‐H., Parsons, M., Nejsum, L. N. Aquaporins differentially regulate cell‐cell adhesion in MDCK cells. FASEB J. 33, 6980–6994 (2019). www.fasebj.org
Traditionally, cutaneous drug delivery is studied by skin accumulation or skin permeation, while alternative techniques may enable the interactions between the drug and the skin to be studied in more ...detail. Time-resolved skin profiling for pharmacokinetic monitoring of two Janus Kinase (JAK) inhibitors, tofacitinib and LEO 37319A, was performed using dermal open-flow microperfusion (dOFM) for sampling of perfusate in an ex vivo and in vivo setup in pig skin. Additionally, matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) was performed to investigate depth-resolved skin distributions at defined time points ex vivo in human skin. By dOFM, higher skin concentrations were observed for tofacitinib compared to LEO 37319A, which was supported by the lower molecular weight, higher solubility, lipophilicity, and degree of protein binding. Using MALDI-MSI, the two compounds were observed to show different skin distributions, which was interpreted to be caused by the difference in the ability of the two molecules to interact with the skin compartments. In conclusion, the techniques assessed time- and depth-resolved skin concentrations and were able to show differences in the pharmacokinetic profiles of two JAK inhibitors. Thus, evidence shows that the two techniques can be used as complementary methods to support decision making in drug development.
Animal and human tissues are used extensively in physiological and pathophysiological research. Due to both ethical considerations and low availability, it is essential to maximize the use of these ...tissues. Therefore, the aim was to develop a new method allowing for multiplex immunofluorescence (IF) staining of kidney sections in order to reuse the same tissue section multiple times. The paraffin‐embedded kidney sections were placed onto coated coverslips and multiplex IF staining was performed. Five rounds of staining were performed where each round consisted of indirect antibody labelling, imaging on a widefield epifluorescence microscope, removal of the antibodies using a stripping buffer, and then re‐staining. In the final round, the tissue was stained with hematoxylin/eosin. Using this method, tubular segments in the nephron, blood vessels, and interstitial cells were labeled. Furthermore, by placing the tissue on coverslips, confocal‐like resolution was obtained using a conventional widefield epifluorescence microscope and a 60x oil objective. Thus, using standard reagents and equipment, paraffin‐embedded tissue was used for multiplex IF staining with increased Z‐resolution. In summary, this method offers time‐saving multiplex IF staining and allows for the retrieval of both quantitative and spatial expressional information of multiple proteins and subsequently for an assessment of the tissue morphology. Due to the simplicity and integrated effectivity of this multiplex IF protocol, it holds the potential to supplement standard IF staining protocols and maximize use of tissue.
PDF
