Purpose Using [18F]PBR06 positron emission tomography (PET) to characterize the time course of stroke-associated neuroinflammation (SAN) in mice, to evaluate whether brain microglia influences motor function after stroke, and to demonstrate the use of [18F]PBR06 PET as a therapeutic assessment tool. Procedures Stroke was induced by transient middle cerebral artery occlusion (MCAO) in Balb/c mice (control, stroke, and stroke with poststroke minocycline treatment). [18 F]PBR06 PET/CT imaging, rotarod tests, and immunohistochemistry (IHC) were performed 3, 11, and 22 days poststroke induction (PSI). Results The stroke group exhibited significantly increased microglial activation, and impaired motor function. Peak microglial activation was 11 days PSI. There was a strong association between microglial activation, motor function, and microglial protein expression on IHC. Minocycline significantly reduced microglial activation and improved motor function by day 22 PSI. Conclusion [18 F]PBR06 PET imaging noninvasively characterizes the time course of SAN, and shows increased microglial activation is associated with decreased motor function.

Background: PSCA, a cell surface protein, is upregulated in many solid tumors & correlates with disease stage. BPX601 is an autologous, T-cell product engineered to contain a PSCA-CD3n CAR plus the small molecule rimiducid (Rim)-inducible MyD88/ CD40 costimulatory domain. BPX601 is optimized for antigen-directed & independent T cell activation, proliferation & persistence, potentially enhancing efficacy in solid tumors versus traditional CARs. This first-in-human study assesses the safety, biological & clinical activity of BPX601 plus Rim in select PSCA-positive cancers. Methods: NCT02744287 is a 2-part, open-label trial. Part 1 is an ongoing 3 þ 3 cell dose escalation to identify the recommended BPX601 cell dose (Day 0) given in combination with a fixed, single Rim dose (0.4 mg/kg; Day 7). Eligibility criteria include previously treated metastatic pancreatic cancer (mPDAC) with measurable disease & positive PSCA expression. Results: Patients received only cyclophosphamide (CTX) for lymphodepletion (LD) within 3 days before BPX601 infusion. Nine adults have been treated across 3 cell dose levels (cells/kg): 1.25x10 6 (cells only), 1.25x10 6 þRim, 2.5x10 6 þRim. All had mPDAC with 2 prior therapies. Common AEs were fatigue & nausea. No DLTs, related SAEs, neurotoxicity or CRS events were reported. Rapid cell engraftment by Day 4 was observed in all patients. No evidence of LD with CTX was seen. Of 6 patients that received Rim: 2 had cell expansion 10-to 20-fold within 7 days; 2 had cell persistence >3 weeks; all had elevated serum cytokines (IP-10, TNFa) correlated with cell expansion. Best response after 1 scan was 4 SD 8 weeks with 2 minor responses (not confirmed; 1 patient had matched CA19-9 decrease) & 2 PD. Disease control without new therapy was 16 & >11 weeks (ongoing) in 1 & 3 patients, respectively. Conclusions: BPX601 with single-dose Rim was well-tolerated & resulted in enhanced T cell expansion & prolonged persistence in some patients despite lack of LD. Evidence of clinical benefit in this heavily pretreated mPDAC population was seen. Part 2 is planned to open soon & will include CTX/fludarabine LD to maximize engraftment as well as gastric & prostate cancers. Clinical trial identification: NCT02744287. Legal entity responsible for the study: Bellicum Pharmaceuticals. Funding: Bellicum Pharmaceuticals.

Tumor-associated macrophages (TAMs) are known to promote physiological processes that drive tumor progression and survival, including angiogenesis, immunosuppression, and invasion. While most studies on TAMs have focused on the molecular mechanisms by which TAMs drive these processes, less focus has been given to direct effects that TAMs may exert on tumor cells. It is known that monocytes are recruited to tumor sites after radiotherapy and support tumor regrowth. In this study we sought to determine if TAMs are able to exert a direct, protective effect on tumor cells against radiotherapy in vitro. We used murine 4T1 cells, a well-characterized animal model for breast cancer, and murine RAW264.7 macrophages to carry out co-culture experiments. Co-culture of 4T1 cells with RAW264.7 macrophages resulted in increased 4T1 cell survival rates post-irradiation compared to 4T1 cells cultured alone. We further determined that this effect is not contact-dependent but mediated through macrophage secreted factors. Next, we tested whether co-culturing RAW264.7 macrophages with 4T1 cells induced macrophage polarization to an M2/TAM phenotype. Using antibodies against M2 markers and FACS, we found that co-culture of RAW macrophages with 4T1 cells polarizes these macrophages toward an M2 phenotype. We hypothesized that the increased 4T1 cell survival rates were mediated by an enhanced DNA damage repair mechanism induced by TAMs. We tested this via a combination of immunofluorescence against phosphorylated histone H2AX and comet assays. We show that co-culture of 4T1 cells with TAMs after irradiation results in lower 4T1 cell levels of DNA damage and a faster decrease in DNA damage over time compared to 4T1 cells alone. Future work will focus on characterizing the molecular mechanism through which TAMs induce this enhanced DNA damage repair response in 4T1 cells after irradiation and we will test whether such response occurs in vivo after radiotherapy. In conclusion, we show that TAMs confer resistance against ionizing radiation to tumor cells in vitro and that this resistance is driven by an enhanced DNA damage repair mechanism in tumor cells induced by TAM secreted factors. Citation Format: Luis A. Soto, Marjan Rafat, Marta Vilalta Colomer, Amato Giaccia, Edward Graves. Tumor-associated macrophages enhance DNA damage repair and improve survival of murine breast cancers after irradiation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1645.

Purpose: To investigate tungsten nanoparticles (WNP) and conventional iodine contrast as potential vehicles for dose enhancers (DE) for dose enhanced radiation therapy (DERT). Methods: We systematically evaluated DERT in silico, in vitro, and in vivo by quantifying dose enhancement, cell kill, and tumor control. Because of variations in the K‐edge and toxicity of these different nanoparticles we expect to see differences in their viability and effective dose boosting. We used Na[M3‐W3(O2)(OAc)a] as our WNP, and iohexol as our iodine agent. Monte Carlo (MC) simulations using the EGSncr codes were performed to quantify the macroscopic dose enhancement as a function of DE concentration from both DEs. The DEs were used in 7, 15, and 30 mg/ml concentrations in vitro with AA8 CHO cell lines and were irradiated with varying doses using a 150 kV beam to establish cell survival curves. Balb‐c mice were injected with the same cell lines used in vitro to grow as subcutaneous tumors. The tumors were directly injected with the DE formulations and a series of fluoroscopic x‐ray images were acquired to determine the biological half‐life of the DEs in the tumor. Mouse tumors with and without DE were irradiated with a 150 kVp beam and tumor regrowth delay was measured. Results: MC simulations showed macroscopic dose enhancement varying from 1.8 to 3.8 for iodine and from 1.9 to 4.0 for tungsten when irradiated with a 150 kV beam. The tungsten in vitro study indicated dose enhancement of a factor of 2.2 for a concentration of 30 mg/mL. The biological half‐life of tungsten in the tumor injected with 200 mg/mL was shown to be 20 minutes. Conclusion: Preliminary in silico, in vitro, and in vivo studies of tungsten nanoparticles and iodine contrast showed the potential for their use in dose enhanced radiation therapy.

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