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The physico-chemical and biological response to conventional and UHDR electron and proton beams was investigated, along with conventional photons. The temporal structure and nature of the beam affected both, with electron beam at ≥1400 Gy/s and proton beam at 0.1 and 1260 Gy/s found to be isoefficient at sparing zebrafish embryos.

Introduction: Cancer cells can escape tumor-specific T cell responses via engagement of inhibitory immune checkpoints. PD-L1/PD-1 interaction is one of the major checkpoints that limit effector T cell function against cancer cells, and monoclonal antibodies that block this interaction have been approved as therapies in multiple tumor indications. As a next generation therapy, small molecule inhibitors of PD-L1 potentially have advantageous properties compared to approved monoclonal antibodies, such as better penetration into solid tumors, reduced immunogenicity, lack of Fc-mediated side effects, convenience of oral administration, and lower cost of goods. We therefore embarked on an effort to identify and develop an orally available small molecule capable of targeting PD-L1/PD-1 interactions. Methods: Inhibition of the PD-L1/PD-1 interaction was measured using a binding assay, followed by a cell culture system assessing PD-1 inhibition of T cell receptor (TCR) activation. Human T cell responses were assessed in vitro using the mixed lymphocyte reaction (MLR) assay, and a human peripheral blood mononuclear cell (PBMC)-mediated tumor cell killing assay. For in vivo studies CCX559 was dosed orally in a syngeneic tumor model and in a human tumor cell/PBMC co-implantation model in immune deficient mice. Results: Using structural information and focused medicinal chemistry, we identified CCX559 as a potent inhibitor of PD-L1 interaction with PD-1. CCX559 prevented PD-L1/PD-1 inhibition of TCR signaling in a cell-based reporter assay, increased IFNγ secretion in allogeneic MLR assays, and increased tumor cell killing by human PBMCs. We demonstrated that CCX559 potentially employs multiple mechanisms to inhibit PD-L1, which are distinct from those published for human anti-PD-L1 antibodies. In murine tumor models, orally administered CCX559 reduced tumor growth similarly to a clinically-approved anti-human PD-L1 antibody. Summary: CCX559 is a highly potent, small molecule PD-L1 inhibitor that can be orally administered. CCX559 enhanced primary human T cell activity in vitro and demonstrated anti-tumor efficacy in two murine tumor models. Based on its unique mechanism of PD-L1 inhibition, strong anti-tumor activity, desirable drug properties, and good safety profile, we plan to advance CCX559 into clinical development in the first half of 2021. Citation Format: Chris Li, Marta Vilalta, Linda S. Ertl, Yu Wang, Yibin Zeng, Pingchen Fan, Christopher Lange, Darren McMurtrie, Ju Yang, Rebecca Lui, Ryan Scamp, Vicky Chhina, Alice Kumamoto, Ryan Ong, Ton Dang, Ashton Easterday, Niky Zhao, Shirley Liu, Rajinder Singh, Israel Charo, Kathleen Sullivan, Thomas J. Schall, Penglie Zhang. CCX559 is a potent orally-administered small molecule PD-L1 inhibitor that induces anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1274.

PurposePreclinical studies of hypoxia are generally done using ectopic xenograft tumors, which behave differently from human tumors. Our previous findings have shown that subcutaneously implanted lung tumors exhibit more hypoxia than their orthotopic implanted or spontaneous K-ras-induced counterparts. We hypothesize that differences in hypoxia are due to site-specific differences in vascularity and perfusion.ProceduresTo compare the presence and functionality of vessels in these tumor models, we studied vascular perfusion in vivo in real time.ResultsOrthotopically implanted and spontaneous K-ras-induced lung tumors showed elevated perfusion, demonstrating vasculature functionality. Little contrast agent uptake was observed within the subcutaneously implanted tumors, indicating vascular dysfunction. These findings were corroborated at the microscopic level with Hoechst 33342 and Meca-32 staining.ConclusionsFrom these observations, we concluded that differences in hypoxia in experimental models is related to vessel perfusion. Thus, appropriate selection of preclinical lung tumor models is essential for the study of hypoxia, angiogenesis and therapies targeting these phenomena.

Introduction: Antibody-based therapies targeting the Programmed cell Death-1/ Programmed Death-Ligand 1 (PD-1/PD-L1) immune checkpoint axis have achieved great success in cancer immunotherapy in recent years. As a next generation therapy, small molecule inhibitors of PD-1/PD-L1 offer the potential for increased tumor penetration, shorter half-life (to better manage immune related adverse events), and lower cost. We therefore embarked on an effort to identify and develop orally available small molecules capable of targeting PD-L1. Methods: We designed and optimized a number of small molecule PD-L1 inhibitors which potently disrupted the interaction of PD-1 with PD-L1. Active compounds were first profiled by an ELISA assay measuring inhibition of the PD-1/PD-L1 interaction, followed by a functional cell-based reporter assay, mixed lymphocyte reaction (MLR) assay, and human peripheral blood mononuclear cell (PBMC)-mediated tumor cell killing assay. Targeted medicinal chemistry efforts were employed to improve potency and oral bioavailability, and candidate compounds were then evaluated in murine tumor models. Due to the specific reactivity of these compounds to human PD-L1 (but not murine PD-L1), a syngeneic tumor model with murine MC-38 colon tumor cells expressing human PD-L1 (MC38-hPD-L1 tumor model) was used. Results: The optimized PD-L1 inhibitors were highly potent in cell-based reporter assay, the MLR assay and PBMC-mediated tumor cell killing assays. These compounds also possess high oral bioavailability and desirable safety profiles. In the MC38-hPD-L1 tumor model, Lead compounds potently reduced tumor growth similarly to an anti-human PD-L1 antibody, which was used as a positive control for the experiments. The tumor microenvironment analysis by flow cytometry demonstrated that these compounds almost completely occupied human PD-L1 on the tumor cells in vivo, and thus could potently block the interaction of PD-1/PD-L1 and enhance the immune responses against tumor. Summary: We have identified and advanced unique small molecule inhibitors of human PD-L1 by rational design and optimization. Molecules resulting from these efforts exhibited marked inhibition of the PD-1/PD-L1 interaction and signaling in vitro, and potent anti-tumor effects in an animal model. Citation Format: Shijie "Chris" Li, Marta Vilalta, Linda S. Ertl, Yu Wang, Yibin Zeng, Pingchen Fan, Christopher Lange, Darren McMurtrie, Ju Yang, Rebecca Lui, Ryan Ong, Vicky Chhina, Alice Kumamoto, Simon Yau, Ton Dang, Ashton Easterday, Shirley Liu, Rajinder Singh, Israel Charo, Thomas J. Schall, Penglie Zhang. Anti-tumor effect of orally available small molecule PD-L1 inhibitors in a murine model of colon adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5693.

Although radiotherapy (RT) decreases the incidence of locoregional recurrence in breast cancer, patients with triple-negative breast cancer (TNBC) have increased risk of local recurrence following breast-conserving therapy. The relationship between RT and local recurrence is unknown. Here, we tested the hypothesis that recurrence in some instances is due to the attraction of circulating tumor cells to irradiated tissues. To evaluate the effect of absolute lymphocyte count on local recurrence after RT in patients with TNBC, we analyzed radiation effects on tumor and immune cell recruitment to tissues in an orthotopic breast cancer model. Recurrent patients exhibited a prolonged low absolute lymphocyte count when compared with nonrecurrent patients following RT. Recruitment of tumor cells to irradiated normal tissues was enhanced in the absence of CD8+ T cells. Macrophages (CD11b+F480+) preceded tumor cell infiltration and were recruited to tissues following RT. Tumor cell recruitment was mitigated by inhibiting macrophage infiltration using maraviroc, an FDA-approved CCR5 receptor antagonist. Our work poses the intriguing possibility that excessive macrophage infiltration in the absence of lymphocytes promotes local recurrence after RT. This combination thus defines a high-risk group of patients with TNBC. Significance: This study establishes the importance of macrophages in driving tumor cell recruitment to sites of local radiation therapy and suggests that this mechanism contributes to local recurrence in women with TNBC that are also immunosuppressed. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/15/4241/F1.large.jpg. Cancer Res; 78(15); 4241–52. ©2018 AACR.

Recently it has been observed in preclinical models that that radiation enhances the recruitment of circulating tumor cells to primary tumors, and results in tumor regrowth after treatment. This process may have implications for clinical radiotherapy, which improves control of a number of tumor types but which, despite continued dose escalation and aggressive fractionation, is unable to fully prevent local recurrences. By irradiating a single tumor within an animal bearing multiple lesions, we observed an increase in tumor cell migration to irradiated and unirradiated sites, suggesting a systemic component to this process. Previous work has identified the cytokine GM-CSF, produced by tumor cells following irradiation, as a key effector of this process. We evaluated the ability of systemic injections of a PEGylated form of GM-CSF to stimulate tumor cell migration. While increases in invasion and migration were observed for tumor cells in a transwell assay, we found that daily injections of PEG-GM-CSF to tumor-bearing animals did not increase migration of cells to tumors, despite the anticipated changes in circulating levels of granulocytes and monocytes produced by this treatment. Combination of PEG-GM-CSF treatment with radiation also did not increase tumor cell migration. These findings suggest that clinical use of GM-CSF to treat neutropenia in cancer patients will not have negative effects on the aggressiveness of residual cancer cells. However, further work is needed to characterize the mechanism by which GM-CSF facilitates systemic recruitment of trafficking tumor cells to tumors.

Purpose: To efficiently translate experimental methods from bench to bedside, it is imperative that laboratory models of cancer mimic human disease as closely as possible. In this study, we sought to compare patterns of hypoxia in several standard and emerging mouse models of lung cancer to establish the appropriateness of each for evaluating the role of oxygen in lung cancer progression and therapeutic response. Experimental Design: Subcutaneous and orthotopic human A549 lung carcinomas growing in nude mice as well as spontaneous K-ras or Myc-induced lung tumors grown in situ or subcutaneously were studied using fluorodeoxyglucose and fluoroazomycin arabinoside positron emission tomography, and postmortem by immunohistochemical observation of the hypoxia marker pimonidazole. The response of these models to the hypoxia-activated cytotoxin PR-104 was also quantified by the formation of γH2AX foci in vitro and in vivo. Finally, our findings were compared with oxygen electrode measurements of human lung cancers. Results: Minimal fluoroazomycin arabinoside and pimonidazole accumulation was seen in tumors growing within the lungs, whereas subcutaneous tumors showed substantial trapping of both hypoxia probes. These observations correlated with the response of these tumors to PR-104, and with the reduced incidence of hypoxia in human lung cancers relative to other solid tumor types. Conclusions: These findings suggest that in situ models of lung cancer in mice may be more reflective of the human disease, and encourage judicious selection of preclinical tumor models for the study of hypoxia imaging and antihypoxic cell therapies. Clin Cancer Res; 16(19); 4843–52. ©2010 AACR.

Mouse CT26 colorectal tumors are heavily infiltrated by tumor-specific CD8 T cells, but nevertheless grow rapidly in Balb/c mice. These tumors are partially responsive to anti-PD-1 (α-PD-1) monoclonal antibody therapy, which suggests that an active suppression of the tumor-specific cytotoxic T cells exists in the untreated tumor. As chemokine receptor 2 (CCR2) is expressed by a potentially suppressive leukocyte subset within these tumors (monocytic myeloid derived suppressor cells aka M-MDSCs), we aimed to test whether CCR2 blockade could enhance the anti-tumor effects of α-PD-1. We have found that the therapeutic effects of α-PD-1 therapy are appreciably enhanced by specific blockade of CCR2 via a small molecule antagonist. This combined α-PD-1/CCR2i approach significantly decreases overall tumor size and increases the proportion of long-term survivors, with more than 50% of the mice (up to 73%) showing complete regression of a previously established tumor. The effects of this combined therapy are dependent on the presence of CD8+ T cells, as tumors do not respond to the therapy in CD8-depleted mice. The anti-CT26 tumor response is specific: long term survivors are resistant to re-inoculation with the CT26 tumor (even without further dosing of either drug) but are not resistant to the 4T1 breast tumor. CCR2 antagonism alters the tumor microenvironment by reducing the number of M-MDSC per gram of tumor (a CCR2hi population phenotypically defined as CD11b+/Ly6G-/Ly6Chi). Reduction in tumor size is inversely proportional to the ratio of CD8 T cells to M-MDSC. These data are consistent with a hypothesis that CCR2 antagonism enhances α-PD-1 therapy by preventing M-MDSC from accumulating within the tumor, thus reducing their suppressive effects on cytotoxic T cells. Citation Format: James J. Campbell, Christine Janson, Linda Ertl, Chris Li, Zhenhua Miao, Antoni Krasinski, Rebecca Lui, Venkat Mali, Jeffrey McMahon, Yibin Zheng, Yu Wang, Xiaoping Zang, Vicky Chhina, Marta Vilalta, Alice Kumamoto, Ton Dang, Shirley Liu, Simon Yao, Penglie Zhang, Thomas J. Schall, Rajinder Singh. Inhibition of chemokine receptor 2 (CCR2) with a small molecule antagonist enhances the effectiveness of checkpoint inhibition by altering the tumor microenvironment in mouse colorectal tumors: Reducing tumor size and increasing long term survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1745.

Breast cancer recurrence remains high in triple-negative cases despite aggressive surgical, radiological, and chemotherapeutic intervention. Recent studies suggest that circulating tumor cell re-seeding of primary tumors may facilitate recurrence. However, the tumor microenvironment's role in recurrence is not well understood. We hypothesize that the irradiated tumor stroma and microenvironment may influence tumor cell migration. In this study, we characterize the effects of normal tissue irradiation on tumor and immune cell migration to evaluate how tumor-stromal interactions modulate recurrence after therapy. This work represents the first step toward elucidating how stromal tissue radiation response contributes to tumor and immune cell recruitment. Mouse embryonic fibroblasts (MEF) were irradiated to 20 Gy with a cesium source. Supernatant was collected after 2 or 7 d incubation to be used as a chemoattractant in a transwell assay to investigate the induction of 4T1 murine or MDA-MB-231 human mammary carcinoma cell invasion. An orthotopic breast cancer model was used to evaluate the effect of radiation on tumor cell migration to normal tissues. Nude mice were inoculated with luciferase labeled 4T1 or MDA-MB-231 cells in the mammary fat pad (MFP), and BALB/c mice with depleted CD4 and CD8+ T cells were inoculated with 4T1 MFP tumors. The contralateral normal MFP was irradiated to a dose of 20 Gy with a 250 kVp cabinet x-ray machine when tumors were palpable. Cell migration was monitored with bioluminescence imaging (BLI) 10 d after irradiation. Irradiated and control tissues were evaluated using immunohistochemistry (IHC). Tissue sections were stained with F4/80 to determine the extent of macrophage infiltration. Flow cytometry was also performed on dissociated irradiated and control tissues to characterize immune cell populations. Of particular interest were CD11b+F4/80+ macrophages and CD11b+GR1+ myeloid-derived suppressor cells (MDSCs). Radiation enhanced tumor cell migration to normal tissues both in vitro and in vivo. 4T1 and MDA-MB-231 cells exhibited an increase in invasion (p This study establishes that normal tissue radiation response may play a role in modulating tumor and immune cell migration after radiation. The increase in macrophage and MDSC infiltration after irradiation indicates the immune contribution in tumor cell migration. These results suggest that the tumor stroma may facilitate tumor cell invasion and tumor regrowth following radiotherapy. Citation Format: Marjan Rafat, Marta Vilalta, Todd A. Aguilera, Amato J. Giaccia, Edward E. Graves. Tumor and immune cell infiltration are enhanced by irradiation of normal tissues in immunocompromised mice. [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 5124.

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.

Skin cancer, which accounts for at least half of all cancers in the United States, is predicted to affects 1 in 5 Americans in their lifetime. Melanoma, the most serious form of skin cancer, is also the leading cause of all skin cancer deaths. This study demonstrates that AKAP12 (SSeCKS, AKAP250, gravin), a hypoxia-inducible scaffold protein, is considerably increased at the protein and RNA level in melanoma cell lines. In addition, there is a significant increase in RNA expression in primary and metastatic tumors when compared to normal specimens, as well as patients with poor prognosis, as measured by diminished 5- year survival. AKAP12 functions to dock many kinases and signaling proteins that regulate cell adhesion, spreading and migration, including: PKC, Src and F-actin. Since these processes are critical for tumor cell metastasis, we hypothesize that AKAP12 could be a central regulator of cancer progression. Stable knockdown of AKAP12 in two melanoma cell lines demonstrate decreases in invasion, migration and clonogenic potential in vitro, as well reduced colony forming abilities in conditions requiring anchorage-independent growth in soft agar. In vivo studies in a metastatic model of melanoma demonstrate a statistically significant decrease in tumors within the lungs of the AKAP12 knockdown groups, highlighting the importance of AKAP12 in metastatic disease. An orthotopic model also demonstrates decreased primary tumor growth and metastatic potential in mice injected with AKAP12 knocked down cells. Additionally, changes in cell morphology characteristic of increased motility and invasiveness are seen in cells containing AKAP12 but not those in which it had been knocked down. While this scaffold protein has been found to act as a tumor suppressor in several tissue types (lung, colon, prostate), we have uncovered its novel oncogenic properties in metastatic melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1460. doi:10.1158/1538-7445.AM2011-1460