Neural stem cell transplantation in patients with progressive multiple sclerosis: an open-label, phase 1 study.

Innovative pro-regenerative treatment strategies for progressive multiple sclerosis (PMS), combining neuroprotection and immunomodulation, represent an unmet need. Neural precursor cells (NPCs) transplanted in animal models of multiple sclerosis have shown preclinical efficacy by promoting neuroprotection and remyelination by releasing molecules sustaining trophic support and neural plasticity. Here we present the results of STEMS, a prospective, therapeutic exploratory, non-randomized, open-label, single-dose-finding phase 1 clinical trial ( NCT03269071 , EudraCT 2016-002020-86), performed at San Raffaele Hospital in Milan, Italy, evaluating the feasibility, safety and tolerability of intrathecally transplanted human fetal NPCs (hfNPCs) in 12 patients with PMS (with evidence of disease progression, Expanded Disability Status Scale ≥6.5, age 18-55 years, disease duration 2-20 years, without any alternative approved therapy). The safety primary outcome was reached, with no severe adverse reactions related to hfNPCs at 2-year follow-up, clearly demonstrating that hfNPC therapy in PMS is feasible, safe and tolerable. Exploratory secondary analyses showed a lower rate of brain atrophy in patients receiving the highest dosage of hfNPCs and increased cerebrospinal fluid levels of anti-inflammatory and neuroprotective molecules. Although preliminary, these results support the rationale and value of future clinical studies with the highest dose of hfNPCs in a larger cohort of patients.

Sleeping Beauty-engineered CAR T cells achieve antileukemic activity without severe toxicities.

BACKGROUNDChimeric antigen receptor (CAR) T cell immunotherapy has resulted in complete remission (CR) and durable response in highly refractory patients. However, logistical complexity and high costs of manufacturing autologous viral products limit CAR T cell availability.METHODSWe report the early results of a phase I/II trial in B cell acute lymphoblastic leukemia (B-ALL) patients relapsed after allogeneic hematopoietic stem cell transplantation (HSCT) using donor-derived CD19 CAR T cells generated with the Sleeping Beauty (SB) transposon and differentiated into cytokine-induced killer (CIK) cells.RESULTSThe cellular product was produced successfully for all patients from the donor peripheral blood (PB) and consisted mostly of CD3+ lymphocytes with 43% CAR expression. Four pediatric and 9 adult patients were infused with a single dose of CAR T cells. Toxicities reported were 2 grade I and 1 grade II cytokine-release syndrome (CRS) cases at the highest dose in the absence of graft-versus-host disease (GVHD), neurotoxicity, or dose-limiting toxicities. Six out of 7 patients receiving the highest doses achieved CR and CR with incomplete blood count recovery (CRi) at day 28. Five out of 6 patients in CR were also minimal residual disease negative (MRD-). Robust expansion was achieved in the majority of the patients. CAR T cells were measurable by transgene copy PCR up to 10 months. Integration site analysis showed a positive safety profile and highly polyclonal repertoire in vitro and at early time points after infusion.CONCLUSIONSB-engineered CAR T cells expand and persist in pediatric and adult B-ALL patients relapsed after HSCT. Antileukemic activity was achieved without severe toxicities.TRIAL REGISTRATIONClinicalTrials.gov NCT03389035.FUNDINGThis study was supported by grants from the Fondazione AIRC per la Ricerca sul Cancro (AIRC); Cancer Research UK (CRUK); the Fundación Científica de la Asociación Española Contra el Cáncer (FC AECC); Ministero Della Salute; Fondazione Regionale per la Ricerca Biomedica (FRRB).

Quantitative assessment of the complex dynamics of G1, S, and G2-M checkpoint activities.

Although studies of cell cycle perturbation and growth inhibition are common practice, they are unable to properly measure the activity of cell cycle checkpoints and frequently convey misinterpretation or incomplete pictures of the response to anticancer treatment. A measure of the strength of the treatment response of all checkpoints, with their time and dose dependence, provides a new way to evaluate the antiproliferative activity of the drugs, fully accounting for variation of the cell fates within a cancer cell line. This is achieved with an interdisciplinary approach, joining information from independent experimental platforms and interpreting all data univocally with a simple mathematical model of cell cycle proliferation. The model connects the dynamics of checkpoint activities at the molecular level with population-based flow cytometric and growth inhibition time course measures. With this method, the response to five drugs, characterized by different molecular mechanisms of action, was studied in a synoptic way, producing a publicly available database of time course measures with different techniques in a range of drug concentrations, from sublethal to frankly cytotoxic. Using the computer simulation program, we were able to closely reproduce all the measures in the experimental database by building for each drug a scenario of the time and dose dependence of G(1), S, and G(2)-M checkpoint activities. We showed that the response to each drug could be described as a combination of a few types of activities, each with its own strength and concentration threshold. The results gained from this method provide a means for exploring new concepts regarding the drug-cell cycle interaction.

Antiproliferative activity of cisplatin detected by CFSE in p53-proficient and p53-deficient cells.

We have previously developed experimental and data analysis procedures to measure the antiproliferative activity of drugs in continuously proliferating cancer cell lines using carboxyfluorescein diacetate succinimidyl ester (CFSE). The method was applied here to analyze the role of p53 in the effect of the anticancer drug cisplatin, distinguishing events occurring in the first generation of cells from those in the second and subsequent generations. A CFSE-loaded colon carcinoma cell line expressing functional wild-type p53 was treated for 1 with cisplatin in parallel with its p53-deficient counterpart, collecting frequency distributions of DNA and CFSE content up to 72 h after treatment. At a sublethal cisplatin concentration proliferation was temporarily inhibited but then the block was overcome and most cells were able to divide several times. The initial block was stronger in HCTp53-/- cells, resulting in a larger proportion of undivided cells at 24 h. This was confirmed and amplified at a higher, lethal concentration, where undivided G(2)M-blocked p53-deficient cells eventually died by non-apoptotic mechanisms, while p53-proficient cells avoided this with a less stringent block. This gave p53-proficient cells more time to repair and eventually decide on survival or apoptotic death before traversing the cycle into their second generation.

The contribution of p53 in the dynamics of cell cycle response to DNA damage interpreted by a mathematical model.

Despite numerous studies on the tumor suppressor p53, a complete picture of its role in cell arrest and killing in G(1), S and G(2)M phases after drug treatment is lacking. We tackled the analysis of the complexity of cell cycle effects combining the time-course measures with different techniques with the aid of a computer program simulating cell cycle progression. This mixed experimental-simulation approach enabled us to decode the dynamics of the cytostatic and cytotoxic responses to cisplatin and doxorubicin treatments in a p53--proficient colon carcinoma cell line (HCT-116) and in its p53-deficient counterpart. We achieved a separate evaluation of the activity of each cell cycle control and we connected these results with measures of p53 level in G(1), S and G(2)M. We confirmed the action of p53 in all cell cycle phases, but also the presence of strong p53-independent cytostatic and cytotoxic activities exerted by both drugs. In G(1) phase, p53 was responsible for a medium/long term block, distinct from the short-term block, which was p53-independent. The delay in traversing S phase was reduced by the presence of p53. In G(2)M phase, despite a strong p53-independent block, there was a weaker but more persistent p53-dependent block. At cytotoxic concentrations, p53-dependent and p53-independent cell death was observed. The former was poorly phase-specific, occurred earlier and exploited the apoptotic mechanism more than p53-independent death. Computer simulation produced a framework where previous partial and sometimes apparently contradictory observations of the p53-mediated effects could be reconciled and explained.

Interpreting cell cycle effects of drugs: the case of melphalan.

Multiple effects usually occur in the cell cycle, during and after the exposure to a drug, while treated cells flowing through the cycle encounter G1, S and G2M checkpoints. We developed a simulation tool connecting the microscopic level of the cellular response in G1, S and G2M with the experimental data of growth inhibition and flow cytometry. We found that multiple-often not intuitive-combinations of cytostatic and cytotoxic effects can be in keeping with the observations. This multiplicity of interpretation can be strongly reduced by considering together data with different methods, ideally reaching a reconstruction of the underlying cell cycle perturbations. Here, we propose an experimental plan including a time course of DNA flow cytometry and absolute cell count measurements with several drug concentrations and a limited number of flow cytometric DNA-Bromodeoxyuridine and TUNEL analyses, coupled with computer simulation. We showed its use in the attempt to define the complete time course of the effects of melphalan on three cancer cell lines. After drug treatment, each subset of cells experienced blocks and lethality in all phases of the cell cycle, but the dynamics was different, the differences being strongly dose-dependent. Our approach allows a better appreciation of the complexity of the cell cycle phenomena associated with drug treatment. It is expected that such level of understanding of the time- and dose-dependence of the cytostatic and cytotoxic effects of a drug might support rational therapeutic design.

Heterogeneous cell response to topotecan in a CFSE-based proliferation test.

BACKGROUND: Carboxyfluorescein diacetate succinimidyl ester (CFSE) is currently used to investigate migration and proliferation of hemopoietic cells. In principle, CFSE is retained by the cells and is shared by the daughter cells at each division, resulting in multimodal flow cytometric CFSE histograms, with each cell generation clustering around half the fluorescence intensity of the previous one. However, intercell variability of CFSE loading results in overlapping peaks, thereby limiting its use with cancer cell lines. METHODS: We used IGROV1 ovarian cancer cells loaded with CFSE at the time of seeding; 24 h later cells were treated with an anticancer drug (topotecan). Potential pitfalls of the analysis were examined, and a procedure of evaluation of CFSE efflux was applied to fix the peak positions with good approximation in advance. Histograms were fitted by a series of gaussians, with each representing cells in a given generation. RESULTS: Effects of topotecan on IGROV1 cells were analyzed in terms of the time course of the percentage of cells that remained undivided or entered the second, third, and subsequent division cycles. A simple algorithm, which combined flow cytometric data with the absolute cell number independently measured by Coulter counter, provided an estimate of the 96-h outcome of the starting cell population by quantifying cells that remained undivided, those able to divide at least once, or those that had died. CONCLUSIONS: We assessed experimental and data analytic procedures for a CFSE-based measurement of antiproliferative activity of drugs in cancer cell lines. A quantitative level was achievable but required a strict procedure for control of the experimental data, which was not straightforward.

Cytostatic and cytotoxic effects of topotecan decoded by a novel mathematical simulation approach.

Topotecan (TPT) is a topoisomerase I inhibitor, and like the other drugs of this family, it is believed to act in a specific way on cells in S phase at the time of treatment. Exploiting a new method, coupling a particular experimental plan with computer simulation, a complete quantitative study of the time dependence and dose dependence of the activity of cell cycle controls has become feasible, and the overall scenario of events after treatment can be reconstructed in detail. We were able to demonstrate that the response of an ovarian cancer cell line to 1 h of treatment with TPT is not limited to inhibition of DNA synthesis, leading to cell death, but involves G(1) and G(2)-M checkpoints. G(1) and G(2)-M block, recycling, and death follow specific dose-dependent kinetics, lasting no less than 3 days after treatment. We also found that cells treated outside S phase contribute significantly to the overall activity. The utility of this analysis was demonstrated by reproducing more complex treatment schemes in which low TPT concentrations were applied for 1 h three times at 24-h intervals. In this case, the simulation clarified the origin of the auto-potentiation observed with repeated 0.2 micro M treatments, in which the cytotoxicity, particularly against S-phase cells, was higher than the cytotoxicity in cells treated with 10 micro M only once. We believe that this approach will help us to understand the complexity and heterogeneity of the response of a cell population to a drug challenge and could help us to establish the rationale for drug scheduling or drug combinations.