Search

Multiple protocols have been published for generation of iMGLs from hESCs/iPSCs. To date, there are no guides to assist researchers to determine the most appropriate methodology for microglial studies. To establish a framework to facilitate future microglial studies, we first performed a comparative transcriptional analysis between iMGLs derived using three published datasets, which allowed us to establish the baseline protocol that is most representative of bona fide human microglia. Secondly, using CRISPR to tag the classic microglial marker CX3CR1 with nanoluciferase and tdTomato, we generated and functionally validated a reporter ESC line. Finally, using this cell line, we demonstrated that co-culture of iMGL precursors with human glia and neurons enhanced transcriptional resemblance of iMGLs to ex vivo microglia. Together, our comprehensive molecular analysis and reporter cell line are a useful resource for neurobiologists seeking to use iMGLs for disease modeling and drug screening studies., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The blood-brain barrier (BBB) presents a barrier for circulating factors, but simultaneously challenges drug delivery. How the BBB is altered in Alzheimer disease (AD) is not fully understood. To facilitate this analysis, we derived brain endothelial cells (iBECs) from human induced pluripotent stem cells (hiPSCs) of several patients carrying the familial AD PSEN1 mutation. We demonstrate that, compared with isogenic PSEN1 corrected and control iBECs, AD-iBECs exhibit altered tight and adherens junction protein expression as well as efflux properties. Furthermore, by applying focused ultrasound (FUS) that transiently opens the BBB and achieves multiple therapeutic effects in AD mouse models, we found an altered permeability to 3-5 kDa dextran as a model cargo and the amyloid-β (Aβ) peptide in AD-iBECs compared with control iBECs. This presents human-derived in vitro models of the BBB as a valuable tool to understand its role and properties in a disease context, with possible implications for drug delivery., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Here we elucidate the effect of Alzheimer disease (AD)-predisposing genetic backgrounds, APOE4, PSEN1ΔE9, and APPswe, on functionality of human microglia-like cells (iMGLs). We present a physiologically relevant high-yield protocol for producing iMGLs from induced pluripotent stem cells. Differentiation is directed with small molecules through primitive erythromyeloid progenitors to re-create microglial ontogeny from yolk sac. The iMGLs express microglial signature genes and respond to ADP with intracellular Ca 2+ release distinguishing them from macrophages. Using 16 iPSC lines from healthy donors, AD patients and isogenic controls, we reveal that the APOE4 genotype has a profound impact on several aspects of microglial functionality, whereas PSEN1ΔE9 and APPswe mutations trigger minor alterations. The APOE4 genotype impairs phagocytosis, migration, and metabolic activity of iMGLs but exacerbates their cytokine secretion. This indicates that APOE4 iMGLs are fundamentally unable to mount normal microglial functionality in AD., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The first US Food and Drug Administration (FDA)- and EMA-approved oncolytic virus has been available since 2015. However, there are no markers available that would predict benefit for the individual patient. During 2007-2012, we treated 290 patients with advanced chemotherapy-refractory cancers, using 10 different oncolytic adenoviruses. Treatments were given in a Finnish Medicines Agency (FIMEA)-regulated individualized patient treatment program (the Advanced Therapy Access Program [ATAP]), which required long-term follow-up of patients, which is presented here. Focusing on the longest surviving patients, some key clinical and biological features are presented as "oncograms." Some key attributes that could be captured in the oncogram are suggested to predict treatment response and survival after oncolytic adenovirus treatment. The oncogram includes immunological laboratory parameters assessed in peripheral blood (leukocytes, neutrophil-to-lymphocyte ratio, interleukin-8 [IL-8], HMGB1, anti-viral neutralizing antibody status), features of the patient (gender, performance status), tumor features (histological tumor type, tumor load, region of metastases), and oncolytic virus-specific features (arming of the virus). The retrospective approach used here facilitates verification in a prospective controlled trial setting. To our knowledge, the oncogram is the first holistic attempt to identify the patients most likely to benefit from adenoviral oncolytic virotherapy.

Alzheimer's disease (AD) is a common neurodegenerative disorder and the leading cause of cognitive impairment. Due to insufficient understanding of the disease mechanisms, there are no efficient therapies for AD. Most studies have focused on neuronal cells, but astrocytes have also been suggested to contribute to AD pathology. We describe here the generation of functional astrocytes from induced pluripotent stem cells (iPSCs) derived from AD patients with PSEN1 ΔE9 mutation, as well as healthy and gene-corrected isogenic controls. AD astrocytes manifest hallmarks of disease pathology, including increased β-amyloid production, altered cytokine release, and dysregulated Ca 2+ homeostasis. Furthermore, due to altered metabolism, AD astrocytes show increased oxidative stress and reduced lactate secretion, as well as compromised neuronal supportive function, as evidenced by altering Ca 2+ transients in healthy neurons. Our results reveal an important role for astrocytes in AD pathology and highlight the strength of iPSC-derived models for brain diseases., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

The development of oncolytic viruses has recently made great progress towards being available to cancer patients. With the breakthrough into clinics, it is crucial to analyze the existing clinical experience and use it as a basis for treatment improvements. Here, we report clinical data from 290 patients treated with oncolytic adenovirus. Using clinical variables and treatment characteristics, we constructed statistical models with regard to treatment response and overall survival (OS). Additionally, we investigated effects of neutralizing antibodies, tumor burden, and peripheral blood leucocyte counts on these outcomes. We found the absence of liver metastases to correlate with an improved rate of disease control (P = 0.021). In multivariate evaluation, patients treated with viruses coding for immunostimulatory granulocyte macrophage colony-stimulating factor were linked to better prognosis (hazard ratio (HR) 0.378, P < 0.001), as well as women with any cancer type (HR 0.694, P = 0.017). In multivariate analysis for imaging response, patients treated via intraperitoneal injection were more likely to achieve disease control (odds ratio (OR) 3.246, P = 0.027). Patients with low neutrophil-to-lymphocyte ratio before treatment had significantly longer OS (P < 0.001). These findings could explain some of the variation seen in treatment outcomes after virotherapy. Furthermore, the results offer hypotheses for treatment optimization and patient selection in oncolytic adenovirus immunotherapy.

Despite many clinical trials conducted with oncolytic viruses, the exact tumor-level mechanisms affecting therapeutic efficacy have not been established. Currently there are no biomarkers available that would predict the clinical outcome to any oncolytic virus. To assess the baseline immunological phenotype and find potential prognostic biomarkers, we monitored mRNA expression levels in 31 tumor biopsy or fluid samples from 27 patients treated with oncolytic adenovirus. Additionally, protein expression was studied from 19 biopsies using immunohistochemical staining. We found highly significant changes in several signaling pathways and genes associated with immune responses, such as B-cell receptor signaling (P < 0.001), granulocyte macrophage colony-stimulating factor (GM-CSF) signaling (P < 0.001), and leukocyte extravasation signaling (P < 0.001), in patients surviving a shorter time than their controls. In immunohistochemical analysis, markers CD4 and CD163 were significantly elevated (P = 0.020 and P = 0.016 respectively), in patients with shorter than expected survival. Interestingly, T-cell exhaustion marker TIM-3 was also found to be significantly upregulated (P = 0.006) in patients with poor prognosis. Collectively, these data suggest that activation of several functions of the innate immunity before treatment is associated with inferior survival in patients treated with oncolytic adenovirus. Conversely, lack of chronic innate inflammation at baseline may predict improved treatment outcome, as suggested by good overall prognosis.

In clinical trials with oncolytic adenoviruses, there has been no mortality associated with treatment vectors. Likewise, in the Advanced Therapy Access Program (ATAP), where 290 patients were treated with 10 different viruses, no vector-related mortality was observed. However, as the patient population who received adenovirus treatments in ATAP represented heavily pretreated patients, often with very advanced disease, some patients died relatively soon after receiving their virus treatment mandating autopsy to investigate cause of death. Eleven such autopsies were performed and confirmed disease progression as the cause of death in each case. The regulatory requirement for investigating the safety of advanced therapy medical products presented a unique opportunity to study tissue samples collected as a routine part of the autopsies. Oncolytic adenoviral DNA was recovered in a wide range of tissues, including injected and noninjected tumors and various normal tissues, demonstrating the ability of the vector to disseminate through the vascular route. Furthermore, we recovered and cultured viable virus from samples of noninjected brain metastases of an intravenously treated patient, confirming that oncolytic adenovirus can reach tumors through the intravascular route. Data presented here give mechanistic insight into mode of action and biodistribution of oncolytic adenoviruses in cancer patients.

The quality of the antitumor immune response is decisive when developing new immunotherapies for cancer. Oncolytic adenoviruses cause a potent immunogenic stimulus and arming them with costimulatory molecules reshapes the immune response further. We evaluated peripheral blood T-cell subsets of 50 patients with refractory solid tumors undergoing treatment with oncolytic adenovirus. These data were compared to changes in antiviral and antitumor T cells, treatment efficacy, overall survival, and T-cell subsets in pre- and post-treatment tumor biopsies. Treatment caused a significant (P < 0.0001) shift in T-cell subsets in blood, characterized by a proportional increase of CD8(+) cells, and decrease of CD4(+) cells. Concomitant treatment with cyclophosphamide and temozolomide resulted in less CD4(+) decrease (P = 0.041) than cyclophosphamide only. Interestingly, we saw a correlation between T-cell changes in peripheral blood and the tumor site. This correlation was positive for CD8(+) and inverse for CD4(+) cells. These findings give insight to the interconnections between peripheral blood and tumor-infiltrating lymphocyte (TIL) populations regarding oncolytic virotherapy. In particular, our data suggest that induction of T-cell response is not sufficient for clinical response in the context of immunosuppressive tumors, and that peripheral blood T cells have a complicated and potentially misleading relationship with TILs.

Oncolytic immunotherapy with cytokine armed replication competent viruses is an emerging approach in cancer treatment. In a recent randomized trial, an increase in response rate was seen but the effect on overall survival is not known with any virus. To facilitate randomized trials, we performed a case-control study assessing the survival of 270 patients treated in an Advanced Therapy Access Program (ATAP), in comparison to matched concurrent controls from the same hospital. The overall survival of all virus treated patients was not increased over controls. However, when analysis was restricted to GMCSF-sensitive tumor types treated with GMSCF-coding viruses, a significant improvement in median survival was present (from 170 to 208 days, P = 0.0012, N = 148). An even larger difference was seen when analysis was restricted to good performance score patients (193 versus 292 days, P = 0.034, N = 90). The survival of ovarian cancer patients was especially promising as median survival nearly quadrupled (P = 0.0003, N = 37). These preliminary data lend support to initiation of randomized clinical trials with GMCSF-coding oncolytic adenoviruses.

Oncolytic adenoviruses and certain chemotherapeutics can induce autophagy and immunogenic cancer cell death. We hypothesized that the combination of oncolytic adenovirus with low-dose temozolomide (TMZ) is safe, effective, and capable of inducing antitumor immune responses. Metronomic low-dose cyclophosphamide (CP) was added to selectively reduce regulatory T-cells. Preclinically, combination therapy inhibited tumor growth, increased autophagy, and triggered immunogenic cell death as indicated by elevated calreticulin, adenosine triphosphate (ATP) release, and nuclear protein high-mobility group box-1 (HMGB1) secretion. A total of 41 combination treatments given to 17 chemotherapy-refractory cancer patients were well tolerated. We observed anti- and proinflammatory cytokine release, evidence of virus replication, and induction of neutralizing antibodies. Tumor cells showed increased autophagy post-treatment. Release of HMGB1 into serum--a possible indicator of immune response--increased in 60% of treatments, and seemed to correlate with tumor-specific T-cell responses, observed in 10/15 cases overall (P = 0.0833). Evidence of antitumor efficacy was seen in 67% of evaluable treatments with a trend for increased survival over matched controls treated with virus only. In summary, the combination of oncolytic adenovirus with low-dose TMZ and metronomic CP increased tumor cell autophagy, elicited antitumor immune responses, and showed promising safety and efficacy.

Patients with advanced solid tumors refractory to and progressing after conventional therapies were treated with three different regimens of low-dose cyclophosphamide (CP) in combination with oncolytic adenovirus. CP was given with oral metronomic dosing (50 mg/day, N = 21), intravenously (single 1,000 mg dose, N = 7) or both (N = 7). Virus was injected intratumorally. Controls (N = 8) received virus without CP. Treatments were well tolerated and safe regardless of schedule. Antibody formation and virus replication were not affected by CP. Metronomic CP (oral and oral + intravenous schedules) decreased regulatory T cells (T(regs)) without compromising induction of antitumor or antiviral T-cell responses. Oncolytic adenovirus given together with metronomic CP increased cytotoxic T cells and induced Th1 type immunity on a systemic level in most patients. All CP regimens resulted in higher rates of disease control than virus only (all P < 0.0001) and the best progression-free (PFS) and overall survival (OS) was seen in the oral + intravenous group. One year PFS and OS were 53 and 42% (P = 0.0016 and P < 0.02 versus virus only), respectively, both which are unusually high for chemotherapy refractory patients. We conclude that low-dose CP results in immunological effects appealing for oncolytic virotherapy. While these first-in-human data suggest good safety, intriguing efficacy and extended survival, the results should be confirmed in a randomized trial.

Augmenting antitumor immunity is a promising way to enhance the potency of oncolytic adenoviral therapy. Granulocyte-macrophage colony-stimulating factor (GMCSF) can mediate antitumor effects by recruiting natural killer cells and by induction of tumor-specific CD8(+) cytotoxic T-lymphocytes. Serotype 5 adenoviruses (Ad5) are commonly used in cancer gene therapy. However, expression of the coxsackie-adenovirus receptor is variable in many advanced tumors and preclinical data have demonstrated an advantage for replacing the Ad5 knob with the Ad3 knob. Here, a 5/3 capsid chimeric and p16-Rb pathway selective oncolytic adenovirus coding for GMCSF was engineered and tested preclinically. A total of 21 patients with advanced solid tumors refractory to standard therapies were then treated intratumorally and intravenously with Ad5/3-D24-GMCSF, which was combined with low-dose metronomic cyclophosphamide to reduce regulatory T cells. No severe adverse events occurred. Analysis of pretreatment samples of malignant pleural effusion and ascites confirmed the efficacy of Ad5/3-D24-GMCSF in transduction and cell killing. Evidence of biological activity of the virus was seen in 13/21 patients and 8/12 showed objective clinical benefit as evaluated by radiology with Response Evaluation Criteria In Solid Tumors (RECIST) criteria. Antiadenoviral and antitumoral immune responses were elicited after treatment. Thus, Ad5/3-D24-GMCSF seems safe in treating cancer patients and promising signs of efficacy were seen.