Image_1_PSMA-Specific CAR-Engineered T Cells for Prostate Cancer: CD28 Outperforms Combined CD28-4-1BB “Super-Stimulation”.jpeg

Prostate cancer (PCa) is the second leading cause of malignancy-related mortality in males in the Western world. Although treatment like prostatectomy and radiotherapy for localized cancer have good results, similar positive outcomes are not achieved in metastatic PCa. Consequently, these aggressive and metastatic forms of PCa urgently need new methods of treatment. We already described an efficient and specific second-generation (2G) Chimeric Antigen Receptor (CAR) against Prostate Specific Membrane Antigen (PSMA), a glycoprotein overexpressed in prostate cancer and also present on neovasculature of several tumor entities. In an attempt to improve efficacy and in vivo survival of anti-PSMA 2G CAR-T cells, we developed a third generation (3G) CAR containing two costimulatory elements, namely CD28 and 4-1BB co-signaling domains, in addition to CD3ζ. Differently from what described for other 3G receptors, our third generation CAR disclosed an antitumor activity in vitro similar to the related 2G CAR that comprises the CD28 co-signaling domain only. Moreover, the additional costimulatory domain produced detrimental effects, which could be attributed to an increased activation-induced cell death (AICD). Indeed, such “superstimulation” resulted in an exhausted phenotype of CAR-T cells, after prolonged in vitro restimulation, a higher frequency of cell death, and an impairment in yielding sufficient numbers of transgenic T lymphocytes. Thus, the optimal combination of costimulatory domains for CAR development should be assessed cautiously and evaluated case-by-case.</p

<i>In vivo</i> therapeutic activity of bioconjugates.

<p>Kaplan-Meier survival curves of mice with peritoneal carcinomatosis from HT-29, MKN-45 or OE-21 tumor cells. Animals were randomly assigned to an experimental group and drug treatment was initiated according to therapeutic schedule reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112240#s2" target="_blank">Materials and Methods</a>. All experimental groups were statistically compared each other, but only significant values are reported in each panel.</p

Assessment of bioconjugate mechanism of action.

<p>A, rearrangement of tumor cell microtubular architecture after drug treatment. HT-29, MKN-45 and OE-21 cells were treated with ONCOFID-P or free paclitaxel for 4 hours at 37°C. After treatment, cells were fixed, permeabilized, and stained with an anti-β-tubulin mAb and anti-mouse Ig Alexa 546-conjugated antiserum. Cells treated with free drug or bioconjugate disclosed the same interferences on the microtubular mesh. B, inhibition of Topo I activity after ONCOFID-S or SN-38 treatment in HT-29, MKN-45 and OE-21 cells. Gels show the supercoiled or relaxed forms of pBR322 plasmid after incubation with a 1∶50 dilution of nuclear protein neat extracts obtained from tumor cells treated with conjugated or free drug for 4 hours. Lane 1, marker; lane 2, relaxed pBR322 plasmid (positive control); lane 3, supercoiled plasmid (negative control); lane 4, supercoiled plasmid in the presence of nuclear protein neat extract from drug-untreated cells; lane 5, supercoiled pBR322 admixed with nuclear protein neat extract from ONCOFID-S treated cells; lane 6, supercoiled pBR322 admixed with nuclear protein neat extract from SN-38-treated cells. C, quantification of the reactions shown in B. Figure reports mean ± SD of 3 independent experiments.</p

Endocytosis pathways involved in bioconjugate cell entry.

<p>HT-29, MKN-45 and OE-21 tumor cells were left untreated (solid line) or treated (dashed line) for 1 hour with selective chemical inhibitors of different pathways involved in endocytosis (amiloride, chlorpromazine, cytochalasin D and filipin III). Subsequently, cells were exposed for 30 minutes to ONCOFID-P and then treated with hyaluronidase for 4 hours, to be finally analyzed by flow cytometry. Data at the upper-right corner of each panel report the respective geo mean values, and the percentage of reduction induced by treatment.</p

Interaction of bioconjugates with cancer cell lines.

<p>A, BODIPY-labeled ONCOFID-P (50 µg/mL in paclitaxel equivalents) or ONCOFID-S (50 µg/mL in SN-38 equivalents) were added to tumor cells and flow cytometry analysis was performed at different time points thereafter (0.5, 1, 2, 5, 10, 15, 30 or 60 minutes). Panels illustrate cytometry profiles at 3 representative time points. B, whole kinetics of interaction at all time points tested. C, kinetics of the fluorescence intensity (geo mean) detected on tumor cells at the same time points analysed as in B. Panels B and C report mean ± SD of 3 independent experiments.</p

Confocal microscopy analysis and co-localization studies.

<p>A, accumulation of bioconjugates in HT-29, MKN-45 and KYSE-30. Cells were incubated with BODIPY-labeled ONCOFID-P (50 µg/mL in paclitaxel equivalents) or ONCOFID-S (50 µg/mL in SN-38 equivalents) for 1 hour, washed and fixed before analysis. B, co-localization analysis of bioconjugates in lysosomes. HT-29, MKN-45 and OE-33 cells were treated with LysoTracker green, incubated with BODIPY-labeled compounds and finally analyzed by confocal microscopy. Left pictures show the fluorescence of the labeled bioconjugates (red) in single cells, while central pictures illustrate signals (green) from lysosomes. The merging of the 2 components is visible in right pictures. Lysosomes were occupied by bioconjugates by ∼90% to 100%, as assessed by the Zeiss’profile software tool. Experiments were repeated at least twice with consistent results.</p

Assessment of <i>in vivo</i> tumor growth and response to therapy.

<p>A, bioluminescence imaging of pharmacologically treated or untreated mice with peritoneal carcinomatosis induced by luciferase-transduced tumors. Panels show three representative mice per group at one month after tumor injection. B, cumulative results. Each box plot reports mean ± SD of total photon emission from 6 mice per group at one month from peritoneal carcinomatosis induction. Statistical analysis (Kruskal-Wallis test) is reported in tables at the right of each panel.</p

Loss of hematopoietic cell-derived oncostatin M worsens diet-induced dysmetabolism in mice

Innate immune cells infiltrate growing adipose and propagate inflammatory clues to metabolically distant tissues, thereby promoting glucose intolerance and insulin resistance. Cytokines of the IL-6 family and gp130 ligands are among such signals. The role played by Oncostatin M (OSM) in the metabolic consequences of overfeeding is debated at least in part because prior studies did not distinguish OSM sources and dynamics. Here, we explored the role of OSM in metabolic responses and used bone marrow transplantation to test the hypothesis that hematopoietic cells are major contributors to the metabolic effects of OSM. We show that OSM is required to adapt during the development of obesity as OSM concentrations are dynamically modulated during high-fat diet (HFD) and Osm-/- mice displayed early-onset glucose intolerance, impaired muscle glucose uptake, worsened liver inflammation and damage. We found that OSM is mostly produced by blood cells, and that deletion of OSM in hematopoietic cells phenocopied glucose intolerance of whole-body Osm-/- mice on HFD, and recapitulated liver damage with increased aminotransferase levels. We thus uncover that modulation of OSM is involved in the metabolic response to overfeeding and that hematopoietic cell-derived OSM can regulate metabolism, likely via multiple effects in different tissues.</p

Assessment of T-body <i>in vivo</i> loco-regional therapeutic efficacy.

<p>(A) Winn Assay. PC3-PIP (<i>left panel</i>) and PC3 (<i>right panel</i>) tumor cells were inoculated s.c. in SCID mice, alone or mixed 1∶1 with T-body-hPSMA/eGFP at opposite flanks of the same animal. Tumor growth was monitored over time by caliper measurement. Number of mice per group, n = 6. (B) Loco-regional therapy. T-body-hPSMA/eGFP at 72 hours post transduction were administered intralesionally and perilesionally in SCID mice 4 days after s.c. injection of PC3-PIP tumor cells (n = 6); untreated animals served as control group (n = 6). <i>Left panel</i> shows tumor volumes, while <i>right panel</i> reports Kaplan-Meyer survival curves of treated and untreated mice. (C) Expression of hPSMA antigen in prostate tumors. PC3-PIP tumor cells from <i>in vitro</i> cultures (first quadrant) or isolated <i>ex-vivo</i> from control or treated mice (second and third quadrant, respectively; dark line) were evaluated for hPSMA expression by flow cytometry. The grey plot corresponds to the isotype control.</p

Assessment of T-body <i>in vivo</i> systemic therapeutic efficacy against disseminated prostate carcinoma.

<p>Rag2^−/−/γc^−/− mice (n = 6) and NOD/SCID mice (n = 6) bearing established bioluminescent PC3-PIP tumors were injected i.v. with 2×10⁷ T-body-hPSMA/eGFP for 3 times at a two day interval. Untreated animals (n = 6 for both mouse strains) were used as controls. (A) Pictures show two representative Rag2^−/−/γc^−/− and NOD/SCID mice (<i>left</i> and <i>right panels</i>, respectively) imaged by BLI at different time points, whereas (B) and (C) graphs report cumulative results of the regions of interest (ROI) in lungs and in total body, respectively. Tumor growth was monitored as photon flux and quantified as photon * sec⁻¹ * cm⁻² * sr⁻¹. Graphs show mean ± SD of three independent experiments. *: P<0.05. The t-Test was used for statistical analysis. (D) Cumulative Kaplan-Meier survival curves of Rag2^−/−/γc^−/− (<i>left panel</i>; untreated mice, black line; median survival = 54 days; treated mice, red line; median survival = 74 days; P = 0.046) and NOD/SCID mice (<i>right panel</i>; untreated mice, black line; median survival = 60 days; treated mice, red line; median survival = not evaluable; P<0.001).</p