bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P6-06-15: Remodelling of calcium influx pathways in breast cancer associated fibroblasts
Authors: Sadras Francisco , Stewart Teneale A , Robitaille Melanie , Peters Amelia A , Kalita-de Croft Priyakshi , Soon Patsy , Saunus Jodi M , Lakhani Sunil R , Roberts-Thomson Sarah J , Monteith Gregory R
Published: March 2020

Disruption of Ca2+ signaling during breast cancer tumorigenesis is now well-established. Despite the increasing recognition of the role of the tumour microenvironment, and cancer associated fibroblasts (CAFs), in tumorigenesis, very few studies have assessed alterations in Ca2+ influx pathways in this context. The aims of this study were to identify changes in the nature of Ca2+ influx in breast CAFs and to define the role of Ca2+ signaling in the induction of CAFs.

We first developed a model for CAF induction using an immortalized human mammary fibroblast cell line, HMF3S. These cells were treated with transforming growth factor beta (TGFβ) to induce a CAF phenotype (HMF3S-CAF) as confirmed by the induction established CAF markers, including alpha smooth muscle actin. Fluorescence Imaging Plate Reader Ca2+ assays demonstrated that store operated Ca2+ entry (SOCE) was reduced as a consequence of TGFβ stimulated CAF induction in HMF3S-CAFs. As a potential mechanism, mRNA levels of the SOCE components, ORAI1, STIM1 and STIM2 were assessed. STIM2 was downregulated and ORAI1 was slightly upregulated suggesting that the decrease in SOCE in HMF3S-CAF cells were independent of mRNA expression changes of these genes.

Voltage gated calcium channel (VGCC) mRNA expression was measured to determine whether other Ca2+ influx pathways were altered during CAF differentiation. Here, we found that Cav 1.2 and Cav 3.2 were significantly upregulated in HMF3S-CAF cells. Assessment of mRNA levels of SOCE components and VGCC in paired normal and CAF patient samples showed no change in SOCE components while, of the VGCCs, only Cav 1.2 and Cav 3.2 were upregulated, suggesting that increased expression of these channels may be a feature of breast cancer CAFs.

The importance of Cav 1.2 and Cav 3.2 in CAF induction was assessed by pharmacological inhibition using nimodipine (Cav 1.2) or ML218 (Cav 3.2), both of which decreased TGFβ-mediated CAF activation. Our preliminary data suggest that siRNA against specific VGCCs may also reduce CAF activation. Our data indicate that Ca2+ signaling is remodelled in CAFs and that VGCC Ca2+ signal is important for breast CAF induction.

Citation Format: Francisco Sadras, Teneale A Stewart, Melanie Robitaille, Amelia A Peters, Priyakshi Kalita-de Croft, Patsy Soon, Jodi M Saunus, Sunil R Lakhani, Sarah J Roberts-Thomson, Gregory R Monteith. Remodelling of calcium influx pathways in breast cancer associated fibroblasts [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-06-15.
Abstract P6-10-28: Therapeutic stratification of triple negative breast cancer by integrating chemosensitivity & phospho-proteome profiles
Authors: Gautam Prson , Qin Xiangju , Vainonen Julia , Nagelli Srikar , Lampe Johanna , Klefström Juha , Westermarck Jukka , Wennerberg Krister , Aittokallio Tero
Published: March 2020

Triple negative breast cancer (TNBC) is a highly aggressive type of breast cancer with poor prognosis that accounts approximately for 15% of breast cancer cases. The lack of targetable hormonal receptors or HER2/ErbB2 makes TNBC therapeutically challenging and existing drug treatments are often ineffective. TNBC is also a highly heterogeneous class of breast cancer, and different genetic approaches have not been so far successful in identifying clinically useful patient stratification strategies to date. In previous studies, we have discovered a correlation between targeted phospho-proteomics and mitotic inhibitor sensitivities in TNBC cells, suggesting the potential of using phospho-proteome profiles to identifying markers predictive of actionable therapeutic strategies. In the current study, we further explored the potential of phospho-proteomics for TNBC stratification and drug chemo-sensitivity prediction. We screened 25 TNBC cell lines against 517 oncology compounds, and in parallel ran mass spectrometry-based global phospho-proteome profiling of the same cell lines, resulting in 4283 confident phospho-peptide quantifications. Using multitask multiple kernel learning, we integrated the phospho-peptide data with other publicly available omics data for the 25 TNBC cell lines (genomics, transcriptomics, metabolomics, and methylomics), and developed a multi-omics machine learning model to quantify the predictive contribution of each omics profile to drug response prediction. When considering all the 517 compounds, we found that the phospho-proteomic and gene expression data were most informative for the prediction of compound’s chemo-sensitivity. This integrated approach has the potential to both identify panels of multi-omics markers for individual drug classes, as well as to stratify the heterogeneous TNBC cells based on their genomic, molecular and functional profiles.

Citation Format: Prson Gautam, Xiangju Qin, Julia Vainonen, Srikar Nagelli, Johanna Lampe, Juha Klefström, Jukka Westermarck, Krister Wennerberg, Tero Aittokallio. Therapeutic stratification of triple negative breast cancer by integrating chemosensitivity & phospho-proteome profiles [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-10-28.
Abstract A1: Clinical outcomes in patients with chronic hepatitis C and metastatic renal cell carcinoma (mRCC) after treatment with nivolumab
Authors: Tsimafeyeu Ilya , Gafanov Rustem , Semenova Anna , Oganesyan Ani , Bondarenko Anastasia , Safina Sufia , Zakurdaeva Kristina , Protsenko Svetlana
Published: March 2020

Clinical trials of immune checkpoint inhibitors have historically excluded patients with chronic hepatitis C (HCV) infection. The safety and efficacy profile of nivolumab is unknown in this under-represented population. Data were collected in a retrospective fashion on 36 patients with metastatic clear-cell mRCC. Patients were required to have a chronic HCV infection, no evidence of other malignancy or cirrhosis, and to have received nivolumab (3 mg/kg every 2 weeks) as second- or third-line therapy. Clinical outcomes included overall survival (OS), progression-free survival (PFS), and objective response rate (ORR). Safety was evaluated. Median follow-up was 17.5 months. Nivolumab was well tolerated in all HCV-infected patients (grade 3-4 toxicity was found in 6 of 36 cases [17%]). No unexpected toxicity was observed. Exposure to nivolumab was not associated with increased risk for hepatocellular carcinoma and cirrhosis (N=0). Medians OS and PFS were 23.9 (95% CI 20.7-26.5) and 6.8 (95% CI 4.9-8.7) months, respectively. 23% of patients had partial responses. The efficacy and safety profile observed in this study supports the administration of nivolumab in mRCC patients infected with HCV and warrants further investigation.

Citation Format: Ilya Tsimafeyeu, Rustem Gafanov, Anna Semenova, Ani Oganesyan, Anastasia Bondarenko, Sufia Safina, Kristina Zakurdaeva, Svetlana Protsenko. Clinical outcomes in patients with chronic hepatitis C and metastatic renal cell carcinoma (mRCC) after treatment with nivolumab [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A1.
Abstract A20: Cancer cell-intrinsic expression of MHCII regulates the immune microenvironment and response to immune therapy in lung adenocarcinoma
Authors: Johnson Amber M. , Bulock Bonnie L. , Neuwelt Alexander J. , Schenk Erin L. , Clambey Eric T. , Nemenoff Raphael A.
Published: March 2020

Immune checkpoint inhibitors targeting PD-1/PD-L1 interactions have shown clinical efficacy in the treatment of multiple cancer, including non-small cell lung cancer (NSCLC). However, the factors determining the extent and duration of therapy are incompletely understood. While antigen presentation via MHC class I interacting with CD8+ T cells has been characterized, the role of MHC class II (MHCII) interacting with CD4+ T cells is less well defined. MHCII expression is usually restricted to antigen-presenting cells, but can be expressed by cancer cells. We examined the effect of cancer cell-intrinsic MHC class II (csMHCII) expression in lung adenocarcinoma on T-cell recruitment to tumors and response to anti-PD-1 therapy. The functional significance of altering csMHCII expression was explored using two orthotopic immunocompetent murine models of non-small cell lung cancer: CMT167 (CMT) and Lewis Lung Carcinoma (LLC). We previously showed that CMT167 tumors are eradicated by anti-PD1 therapy, while LLC tumors are resistant. RNA-seq analysis of cancer cells recovered from tumors revealed that csMHCII correlated with response to anti-PD1 therapy, with immunotherapy-sensitive CMT167 cells being csMHCII positive, while resistant LLC cells were csMHCII negative. To test the functional effects of csMHCII, MHCII expression was altered on the cancer cells through loss and gain of function of CIITA, a master regulator of the MHCII pathway. Loss of CIITA in CMT167 decreased csMHCII and converted tumors from immunotherapy sensitive to immunotherapy resistant. This was associated with decreased T-cell infiltration and lower levels of Th1 cytokines. Conversely, overexpression of CIITA in LLC cells resulted in csMHCII in vitro and in vivo. Enforced expression of CIITA increased T-cell infiltration and sensitized tumors to anti-PD-1 therapy. csMHCII expression was also examined in a subset of surgically resected human lung adenocarcinomas by multispectral imaging, and positively correlated with T-cell infiltration. These studies demonstrate a functional role for csMHCII in regulating T-cell infiltration and sensitivity to anti-PD-1.

Citation Format: Amber M. Johnson, Bonnie L. Bulock, Alexander J. Neuwelt, Erin L. Schenk, Eric T. Clambey, Raphael A. Nemenoff. Cancer cell-intrinsic expression of MHCII regulates the immune microenvironment and response to immune therapy in lung adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A20.
Abstract A28: A comparison of a DNA-sourced neoantigen vaccine to an RNA-sourced frameshift vaccine in the mouse ovarian cancer model
Authors: Batista Milene Tavares , Murphy Sierra , Lainson John , Shen Lui , Johnston Stephen
Published: March 2020

Development of cancer vaccines is currently focused on using neoantigens arising from mutations in the DNA of tumors. We have proposed, and demonstrated, that errors in RNA processing that create frameshift (FS) neoantigens are also a good source of vaccine components, even in tumors that are DNA mutation poor. Here we directly compare the two vaccine approaches in the mouse ovarian model, ID8. Martin et al. (2016) reported that 7 DNA neoepitopes in the ovarian tumor line (ID8-G7) failed to show any protection when tested individually as peptide vaccines even though the peptides elicited an immune response. We created a pooled peptide vaccine consisting of these 7 neoantigens (NeoAg vax). We compared this vaccine to one composed of 13 RNA-sourced FS neoantigens (FS vax). These FSs had conferred protection when tested in the 4T1 mammary and/or B16 melanoma models. 10 of the FSs arise from mis-transcription thru microsatellites and 3 from mis-splicing of exons. In a prophylactic vaccination protocol, both vaccines induce ~20% extended survival compared to mock controls. The FS vax elicited higher numbers of ELISpots than the ID8 control while the NeoAg vax did not when splenocytes were peptide stimulated. The FS vax and NeoAg vax induced T-cells were only significantly cytotoxic to the ID8 cells if anti-PD-L1/CTLA-4 antibodies were included. In the therapeutic vaccination protocol, both the FS vax and the NeoAg vax conferred extended survival in ~20% of the mice compared to controls. Neither vaccine induced more ELISpots compared to controls using peptides. However, the FS vax did elicit more ELISpots in response to ID8 stimulation and more ID8 cytotoxicity than the NeoAg vax. From this work we first conclude that the DNA-sourced neoantigens that did not protect individually can when pooled. Secondly, and more importantly, the RNA-sourced, FS neoantigens can perform as well as the DNA-sourced neoantigens, at least in this model. This may be important as we have shown that all tumors surveyed to date, in contrast to DNA neoantigens, have abundant RNA-sourced FS neoantigens.

Citation Format: Milene Tavares Batista, Sierra Murphy, John Lainson, Lui Shen, Stephen Johnston. A comparison of a DNA-sourced neoantigen vaccine to an RNA-sourced frameshift vaccine in the mouse ovarian cancer model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A28.
Abstract A31: High aneuploidy levels are linked to a reduced immune-cell abundance in metastatic castrate-resistant prostate cancer
Authors: Vidotto Thiago , Salles Daniela C. , Lotan Tamara L.
Published: March 2020

Single-agent immunotherapeutic approaches are promising for treating castrate-resistant prostate cancer (CRPC) patients; however, to date no significant improvement in survival has been reported. Mixed response to therapy occurs because metastatic lesions are highly heterogeneous. These lesions exhibit distinct genomic changes that are linked to a tumor microenvironment that is often poorly populated by immune cells. In other tumor types, a high percent genome altered (e.g., aneuploidy) associates with an overall reduction in immune response in the primary tumor microenvironment. However, the association between genomic changes and immune cell composition is poorly understood in metastatic prostate cancers. Thus, we quantified the abundance of 22 immune cell types in 330 tumors from two independent public domain CRPC cohorts and 491 primary prostate tumors from TCGA using the Absolute Mode from CIBERSORT. The estimation in CRPC was performed using whole-transcriptome data from West Coast Dream Team (Abida et al., 2019; n=212) and East Coast Dream Team (Robinson et al., 2015; n=117) samples. We did not identify any significant correlations between mutational load and immune cell abundance in the three investigated cohorts. In primary tumors, aneuploidy was weakly negatively correlated with CD8+ and CD4+ T-cell abundance (R=-0.14 and R=-0.09, P<0.05). Overall, metastatic lesions demonstrated a low absolute abundance of the majority of immune cell subtypes, with higher levels of CD8+ T-cells, memory CD4+ T-cells, macrophages, and plasma cells. We found that bone lesions had the lowest abundance of CD8+ T-cells (Kruskal-Wallis, p=0.05) and the highest abundance of macrophages (P<0.0001) compared to lymph node and liver samples. Overall, aneuploidy was negatively correlated with immune cell abundance in both metastatic cohorts (Spearman correlation, P<0.05). Interestingly, lymph node metastases showed the strongest negative correlation between aneuploidy and T-cell abundance in both West and East cohorts (R=-0.36 and R=-0.32, respectively, p<0.01). Our preliminary results show that the extent of chromosomal aberrations in CRPC is significantly associated with underlying immune cell infiltrates. These results have potential implications for future immunotherapy trials in prostate cancer.

Citation Format: Thiago Vidotto, Daniela C. Salles, Tamara L. Lotan. High aneuploidy levels are linked to a reduced immune-cell abundance in metastatic castrate-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A31.
Abstract A52: Systemic anti-PD-1 immunotherapy results in PD-1 blockade on T cells in the cerebrospinal fluid
Authors: Wang Dongrui , Portnow Jana , Tran Vivi , Chiu Vivian , Brito Alfonso , Alizadeh Darya , Starr Renate , Kilpatrick Julie , McNamara Paige , Forman Stephen J. , Badie Behnam , Synold Timothy W. , Brown Christine E.
Published: March 2020

Immune checkpoint inhibitors have shown promising clinical efficacy in many types of cancers, including some with metastasis into the central neural system (CNS). However, little is known about the CNS penetration of immune checkpoint inhibitors and their bioactivity on the locoregional T cells. Here, we analyzed the CNS and systemic concentration of pembrolizumab (Pembro) in high-grade glioma patients, who were concurrently treated with intravenously administered Pembro and locoregionally delivered chimeric antigen receptor (CAR) T cells. A total of 99 paired cerebrospinal fluid (CSF) and serum samples were collected from 10 patients during multicycle treatment of CAR T cell and Pembro. We discovered that after the first Pembro infusion, CSF antibody concentrations were increased slower than serum concentrations. However, CSF antibody concentrations reached steady-state 24 hours after Pembro infusion and were maintained stable over the 21-day infusion cycles. Although average CSF Pembro concentrations (3.2nM) were only 1% of serum levels (329nM), we observed effective PD-1 blockade on the T cells in CSF samples of all analyzed patients. The blocking effect was found on both endogenous T cells and locoregionally administered CAR T cells. Further, PD-1 on CD3/CD28 beads-activated T cells was completely blocked after incubation with cell-depleted CSF samples, suggesting the bioactivity of Pembro that penetrated into the CSF. Using a dose-titration assay, we also confirmed that CSF Pembro concentrations were sufficient to ameliorate the cytotoxic activity of CAR T cells against repetitive tumor challenges. Together, for the first time, we showed the dynamic Pembro levels in the CSF throughout multiple treatment cycles and confirmed that systemically administered immune checkpoint inhibitors can penetrate into the CNS and elicit blocking effect on T cells. Our findings add strength to the rationale for combining immune checkpoint inhibitors with locoregionally delivered CAR T cells and other types of immunotherapy for the treatment of brain tumors.

Citation Format: Dongrui Wang, Jana Portnow, Vivi Tran, Vivian Chiu, Alfonso Brito, Darya Alizadeh, Renate Starr, Julie Kilpatrick, Paige McNamara, Stephen J. Forman, Behnam Badie, Timothy W. Synold, Christine E. Brown. Systemic anti-PD-1 immunotherapy results in PD-1 blockade on T cells in the cerebrospinal fluid [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A52.
Abstract A55: Uncovering the mechanism of Trib1 in cancer immunotherapy
Authors: Iheanacho Franklin , Stein Sarah J. , Rome Kelly S. , Jordan Martha S. , Pear Warren S.
Published: March 2020

T cells provide a fundamental role in cell-mediated immunity, but during chronic antigen exposure such as in the case of cancer, T cells begin to lose effector function and undergo exhaustion. Our goal is to find a target within T cells that could be manipulated to prolong T-cell function and reduce tumor growth. Initial experiments focused on the pseudokinase Trib1, as data suggest that Trib1 acts as a negative regulator of T-cell activation. T cells isolated from mice in which Trib1 was specifically ablated within the T-cell compartment produced more IL-2 compared to wild-type T cells. T cells lacking Trib1 also proliferated more than wild-type T cells. In mice lacking Trib1 in grafted tumor models, tumor growth was reduced. These observations suggest that Trib1 restricts T-cell activation and function. Trib1 is a pseudokinase that is thought to act as a scaffolding molecule to facilitate protein interactions. To understand the mechanism by which Trib1 regulates T-cell function, we performed a proteomic screen to identify interacting partners. The screen was performed in Jurkat cells, an immortalized human T-cell line, expressing a FLAG/strep tagged Trib1. A FLAG pulldown was performed to isolate Trib1, and interacting partners were identified by mass spectrometry. The screen revealed two interacting partners: RFWD2, an E3 ubiquitin ligase also known as COP1, and DET1, a binding partner of COP1. These results provide evidence that Trib1 interacts with COP1 in T cells and suggest that this interaction might be important in the regulation of T-cell function and the regression of tumor growth.

Note:This abstract was not presented at the conference.

Citation Format: Franklin Iheanacho, Sarah J. Stein, Kelly S. Rome, Martha S. Jordan, Warren S. Pear. Uncovering the mechanism of Trib1 in cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A55.
Abstract A61: Human leukocyte antigen G as a novel target for switch-based chimeric antigen receptor natural killer cell therapy of solid cancers
Authors: Jan Chia-Ing , Huang Shi-Wei , Canoll Peter , Lin Yu-Chuan , Lu Hsin-Man , Chio Shao-Chih , Cho Der-Yang
Published: March 2020

Human leukocyte antigen G (HLA-G) is a molecule within the tumor microenvironment (TME) that modulates the innate and adaptive immune systems by interacting with inhibitory receptors on the surface of immune cells and thus functions as an immune checkpoint. It could be potentially expressed by all tumor types but is not expressed by either healthy tissues surrounding the tumor cells or vital normal tissues. Chimeric antigen receptors (CARs) for adoptive cell therapy (ACT) have been successful in clinical trials against hematologic cancers; however, challenges have been encountered when applying this approach to the treatment of solid tumors. These obstacles are mainly due to the lack of a ubiquitous tumor-associated antigen (TAA) across different tumor types without “on-target/off-tumor” reactivity and the immunosuppressive nonphysical TME. To address these issues, we developed a novel switch HLA-G CAR carrying an inducible Caspase9 (iC9) suicide gene system that binds to HLA-G1~G7 isoforms and expressed this CAR in natural killer (NK) cells. We tested these HLA-G CAR-NK cells in a variety of adult cancer models and discovered that they mediate significant tumor cytolysis in triple-negative breast cancer (TNBC), glioblastoma (GBM), pancreatic (PA) cancer, and ovarian (OV) cancer in vitro and in TNBC and GBM xenograft models in vivo. Coculturing HLA-G CAR-NK cells with vital normal cell lines did not cause cell damage. We further discovered that surface-exposed HLA-G is chemoinducible, which in turn increases tumor sensitivity to both HLA-G CAR effector-mediated antitumor responses and tumor-infiltrative NK cells. The underlying mechanism of tumor and HLA-G CAR NK cell interaction may be through upregulation of membranous HLA-G via demethylation of the TAP-1 promoter or enhanced activity of the TAP1/signal peptide peptidase (SPP) pathways. In conclusion, HLA-G CAR-NK cells could be an option for treating solid tumors of different cell types, and a regimen comprising chemotherapy followed by HLA-G CAR immunotherapy may improve responses compared to those achieved with CAR-T cell immunotherapy alone.

Citation Format: Chia-Ing Jan, Shi-Wei Huang, Peter Canoll, Yu-Chuan Lin, Hsin-Man Lu, Shao-Chih Chio, Der-Yang Cho. Human leukocyte antigen G as a novel target for switch-based chimeric antigen receptor natural killer cell therapy of solid cancers [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A61.
Abstract A77: Antibody targeting tumor-derived soluble NKG2D ligand sMIC provides dual costimulation of CD8 T cells and enables sMIC+ tumors to respond to PD1/PD-L1 blockade therapy
Authors: Zhang Jinyu , Larrocha Pablo , Dhar Payal , Wu Jennifer
Published: March 2020

Background: Insufficient costimulation accounts for a great deal of the suboptimal activation of cytotoxic CD8 T cells (CTLs) and presumably unsatisfactory clinical expectation of PD1/PDL1 therapy. Tumor-derived soluble NKG2D ligands are associated with poor clinical response to PD1/PDL1 blockade therapy in cancer patients. One of the most frequently occurring tumor-derived soluble NKG2D ligands, the soluble MHC I chain-related molecule (sMIC), can impair costimulation to CD8 T cells. We investigated whether cotargeting sMIC can provide optimal costimulation to CTLs and enhance the therapeutic effect of PD1/PDL1 blockades.

Methods: Single-agent therapy of a PD1/PDL1 blockade antibody or a sMIC-targeting non-blocking antibody or a combination therapy of the two antibodies were applied to well-characterized preclinical MIC/sMIC+ tumor models that closely resemble the NKG2D-mediated oncoimmune dynamics of MIC+ cancer patients. Therapeutic efficacy and associated effector mechanisms were evaluated.

Results: We show that antibody cotargeting sMIC enables or enhances the response of sMIC+ tumors to PD1/PD-L1 blockade therapy. The therapy response of the combination therapy was associated with enhanced antigen-specific CD8 T-cell enrichment and function in tumors. We show that cotargeting sMIC with a nonblocking antibody provides antigen-specific CD8 T cells with NKG2D and CD28 dual costimulation, in addition to elimination of inhibitory signals, and thus amplifies antigen-specific CD8 T-cell antitumor responses.

Conclusion: Our findings provide the proof-of-concept rationale and previously undiscovered mechanisms for cotargeting sMIC to enable and enhance the response to PD1/PDL1 blockade therapy in sMIC+ cancer patients.

Citation Format: Jinyu Zhang, Pablo Larrocha, Payal Dhar, Jennifer Wu. Antibody targeting tumor-derived soluble NKG2D ligand sMIC provides dual costimulation of CD8 T cells and enables sMIC+ tumors to respond to PD1/PD-L1 blockade therapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A77.
Abstract A78: Preclinical evaluation of 3D185, a novel potent inhibitor of FGFR1/2/3 and CSF-1R, in FGFR-dependent and macrophage-dominant cancer models
Authors: Dai Yang , Ji Yichun , Shen Yanyan , Su Yi , Liu Bo , Wang Yueliang , Sun Deqiao , Jiang Yuchen , Zha Chuantao , Xie Zuoquan , Ding Jian , Geng Meiyu , Ai Jing , Peng Xia , Hou Pengcong , Chen Yi
Published: March 2020

The interaction between tumor cells and their immunosuppressive microenvironment promotes tumor progression and drug resistance. Thus, simultaneously targeting tumor cells and stromal cells is expected to have synergistic antitumor effects. Herein, we present the preclinical antitumor investigation of 3D185, a novel dual inhibitor targeting FGFRs, which are oncogenic drivers, and CSF-1R, which is the major survival factor for protumor macrophages. The antitumor characteristics of 3D185 were assessed by a range of assays, including kinase profiling, cell viability, cell migration, immunoblotting, CD8+ T-cell suppression, and in vivo antitumor efficacy, followed by flow cytometric and immunohistochemical analyses of tumor-infiltrating immune cells and endothelial cells in nude mice and immune-competent mice. We found that 3D185 significantly inhibited the kinase activity of FGFR1/2/3 and CSF-1R, with equal potency and high selectivity over other kinases. 3D185 suppressed FGFR signaling and tumor cell growth in FGFR-driven models both in vitro and in vivo. In addition, 3D185 could inhibit the survival and M2-like polarization of protumor macrophages, reversing the immunosuppressive effect of macrophages on CD8+ T cells as well as CSF1-differentiated macrophage induced-FGFR3-aberrant cancer cell migration. Furthermore, 3D185 inhibited tumor growth via remodeling the tumor microenvironment in TAM-dominated tumor models. In summary, 3D185 is a dual inhibitor against FGFR1/2/3 and CSF-1R that exhibits potent antitumor activities. This compound is promising because it simultaneously targets tumor cells per se and the immunosuppressive tumor microenvironment to synergistically antagonize tumors. Our study provides a solid foundation for the investigation of 3D185 in cancer patients, particularly in patients with aberrant FGFR and abundant macrophages, who respond poorly to classic pan-FGFRi treatment. Now 3D185 is in phase I clinical trials.

Citation Format: Yang Dai, Yichun Ji, Yanyan Shen, Yi Su, Bo Liu, Yueliang Wang, Deqiao Sun, Yuchen Jiang, Chuantao Zha, Zuoquan Xie, Jian Ding, Meiyu Geng, Jing Ai, Xia Peng, Pengcong Hou, Yi Chen. Preclinical evaluation of 3D185, a novel potent inhibitor of FGFR1/2/3 and CSF-1R, in FGFR-dependent and macrophage-dominant cancer models [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A78.
Abstract A91: T-cell regulation in pancreatic ductal adenocarcinoma
Authors: Sivakumar Shivan , Abu-Shah Enas , Ahern David , Mangal Nagina , Reddy Srikanth , Rendek Aniko , Soonawalla Zahir , Silva Michael , Middleton Mark , Dustin Michael L.
Published: March 2020

Background: Pancreatic cancer has the worst prognosis of any human malignancy. We have shown using transcriptomics and histopathology that immune infiltrate in resection samples from a Whipple’s operation is predictive of prognosis. In particular, lymphocytes appear to be a major prognostic marker. With promising immunotherapies being proposed for other cancers, there is a need for a deeper understanding of the immune landscape of pancreatic cancer to identify points of intervention.

Methods: We have developed a 37-marker mass cytometry staining panel to characterize the dominant immune populations within primary pancreatic cancer. Our panel further analyzes T-cell subpopulations and their functional status, including a host of clinically relevant checkpoint markers and immunosuppressive signatures.

Results: The degree of immune infiltration we observe is highly variable between patients, but all patients equivocally show a complex immune microenvironment consistent of macrophages, neutrophils, and different lymphocytes. The T cells infiltrating the tumor, both CD4 and CD8 T cells, appear to be dysfunctional with hardly any activation signature. A highly suppressive phenotype also characterizes the regulatory T-cell population. Our data suggest that the microenvironment of pancreatic cancer is extremely suppressive and could be a major driver of poor prognosis. Yet, this work identifies potential therapeutic targets and avenues that should be further investigated and may inspire future clinical trials.

Future Work: We are planning to investigate the behavior of T-regs from PDAC using in vitro assays. To that end, we will use a recently developed advanced 3D culture system to visualize their interactions with CD8s and the functional consequences of those interactions on CD8-mediated killing.

Citation Format: Shivan Sivakumar, Enas Abu-Shah, David Ahern, Nagina Mangal, Srikanth Reddy, Aniko Rendek, Zahir Soonawalla, Michael Silva, Mark Middleton, Michael L. Dustin. T-cell regulation in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A91.
Abstract A92: Analysis of lung-resident macrophages as potential regulators of disseminated tumor cell fate
Authors: Dalla Erica , Casanova-Acebes Maria , Hung Margaret , Linde Nina , Merad Miriam , Aguirre-Ghiso Julio
Published: March 2020

The majority of cancer-related deaths are due to metastasis at secondary organs originating from solitary disseminated tumor cells (DTCs), which can remain dormant for several years. In the lung, some DTCs undergo a period of dormancy before proliferation to form metastasis, while others will not grow into metastatic lesions. As macrophages are key immune components of the microenvironment at distant sites, we aim to understand their potential role in regulating DTC dormancy and awakening. To test the contribution of lung macrophages to cancer cell proliferation, we first compared the effect of lung-resident alveolar macrophages (AMs) and bone marrow-derived monocytes (BM MOs) on cancer cell phenotypes. Our results indicate that AMs may suppress growth and induce EMT (CDH1lo/TWIST1hi phenotype) in cancer cells (of both early and late stages), while BM MOs promote growth. We further assessed the effect of these distinct macrophage populations on survival and proliferation of single cancer cells in a 3D matrigel assay to mimic single DTCs in the lung. Here, AMs appear to retain cancer cells as single cells, while BM MOs favor a switch to proliferation and colony growth. We further used conditioned media (CM) from lung cultures from naive and macrophage-depleted animals to study how organ-specific cues from macrophages affected tumor cell growth. We performed a proteomic analysis of the lung secretome and identified DKK3 as differentially produced before and after macrophage depletion. DKK3, a regulator of Wnt signaling, was absent in the depleted condition. We then tested its effect on cell proliferation in mammary cancer cell sphere formation assays, and we observed that DKK3 increased sphere formation and growth of early- and late-stage cells, respectively. We will next test whether DKK3 production is derived from AMs, BM MOs, or other lung resident cells and we will profile AMs and BM MOs from early and late stages of tumor progression to determine their gene expression profiles at different times of DTC colonization. To explore the role of AMs in dormancy in vivo, we will perform AM depletion in the CD169-DTR animal model. Here, we will deplete specifically tissue-resident AMs and determine DTC fate after tail-vein injection of breast cancer cells. Our study will thus allow us to determine the contribution of different macrophage populations to metastatic dormancy.

Citation Format: Erica Dalla, Maria Casanova-Acebes, Margaret Hung, Nina Linde, Miriam Merad, Julio Aguirre-Ghiso. Analysis of lung-resident macrophages as potential regulators of disseminated tumor cell fate [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A92.
Abstract A97: Neutrophil extracellular traps (NETs) promote immune escape and metastatic growth after surgical stress
Authors: Yazdani Hamza O. , Kaltenmeier Christof T. , Geller David , Tohme Samer
Published: March 2020

Introduction: Surgery induces local and systemic inflammatory and immunosuppressive disturbances that correlate with adverse cancer outcomes. Following surgical stress, NETs promote the growth of liver micrometastases. We hypothesize that NETs support metastatic growth by promoting immune escape.

Methods: Liver ischemia reperfusion was performed 5 days after a murine liver metastatic model was established.

Results: Liver PD-L1 expression increased and peaked between 6-48 hours after reperfusion. A smaller peak of PD-L1 was observed after 2 weeks as the metastatic tumors grew. During the first 48 hours, the predominant source of PD-L1 was CD45+-CD11b+-Ly6G+ cells. PD-L1 expression was significantly decreased when NETs were inhibited either genetically by using PAD4KO mice or DNAse injections. In vitro, we confirmed both human and murine NETs express high levels of PD-L1. After liver I/R, tumor-infiltrating T cells expressed inhibitory receptors and were highly dysfunctional with reduced cytokine production and proliferative capacity in the NET-rich tumors compared to tumors from sham mice. T-cell exhaustion was reversed in mice treated with DNAse or PAD4KO mice. Similar results were observed in vitro as NETs suppressed T-cell function via PDL-1/PD-1 interaction. Treating mice with anti-PD-L1 during the first 48 hours after reperfusion reversed T-cell exhaustion and decreased tumor growth.

Conclusion: NETs induced by surgical stress cause immune escape by secreting PD-L1, increasing T-cell exhaustion, and upregulation of immunosuppressive ligands on tumor cells. These findings suggest avenues for overcoming the perioperative accelerated tumor growth and immunosuppressive state and ultimately improving long-term oncologic outcomes.

Citation Format: Hamza O. Yazdani, Christof T. Kaltenmeier, David Geller, Samer Tohme. Neutrophil extracellular traps (NETs) promote immune escape and metastatic growth after surgical stress [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A97.
Abstract B100: An emerging model for cancer development from a tumor microenvironment perspective in mice and humans
Authors: Yamaguchi Ryuji , Perkins Guy
Published: March 2020

Introduction: We constructed a phylogenic tree of TME based on the transcriptomes from melanoma biopsy samples. From these data, we developed a model that fits the clinical observations in various solid cancer. However, clinical data on the development of TME are like snapshots of cancer at discrete points and thus represent partial views of a dynamic process. It is a model based on an inductive argument. To make the argument more deductive, we examined whether the model fits observations of murine cancer progression. There are only a few dozen mouse cancers in which cancer development takes place reliably and reproducibly in mice with intact immune system. These cancer models in mice augmented the developmental model of TME based on the clinical data.

Summary: The tumor development is initiated by a slowly growing, nearly homogeneous colony of cancer cells that can evade detection by the cells’ innate mechanism of immunity such as natural killer (NK) cells (1st Stage; Colonization). Subsequently, the colony develops into a tumor filled with lymphocytes and stromal cells, releasing proinflammatory cytokines, growth factors, and chemokines (2nd Stage; Lymphocyte Infiltration). Cancer progression proceeds to a well-vesiculated silent tumor releasing no inflammatory signal, being nearly devoid of lymphocytes (3rd Stage; Silenced). Eventually some cancer cells within a tumor undertake epithelial-to-mesenchymal transition (EMT), which leads to cancer metastasis (4th Stage; EMT). If a circulating metastasized cancer cell finds a niche in a new tissue and evades detection by NK cells, it can establish a new colony in which very few stromal cells are present (5th Stage; Metastasis), which is much like a colony at the first stage of development. At every stage, cancer cells influence their own TME and in turn, the TME influences the cancer cells contained within, either by direct interaction between cancer cells and stromal cells or through exchange of cytokines.

Conclusion: There seem to be aspects of cancer development that are independent of cell types and DNA mutations. The hypothesis is that the aspects that are governed solely by the TME can be discovered. Therefore, a productive approach would be to study a particular solid cancer and learn when and how a tumor recruits lymphocytes, fibroblasts, and macrophages to build a Stage 2 environment, and when and how the environment is changed into Stage 3 and Stage 4, and then test how chemotherapeutics and immunotherapies would affect these environments. It is anticipated that most of the knowledge gained would be applicable to essentially any other solid cancer.

Citation Format: Ryuji Yamaguchi, Guy Perkins. An emerging model for cancer development from a tumor microenvironment perspective in mice and humans [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B100.
Abstract B102: Epithelium-derived cytokines in breast carcinogenesis
Authors: Kroon Sanne , Dempsey Kaitlin , Boieri Margherita , Demehri Shawn
Published: March 2020

Novel approaches in cancer immunotherapy have led to effective therapeutics against late-stage cancers. However, the role of the immune system in protection against early-stage cancers remains uncertain. We have previously discovered that thymic stromal lymphopoietin (TSLP) blocks early stages of breast cancer development through activation of CD4+ T cells. TSLP is an epithelium-derived danger signal that plays a key role in initiating an immune response upon cell damage and stress. The aim of this study is to investigate the potential involvement of other epithelial-derived cytokines in the activation of an immune response against premalignant breast lesions. To identify these signals, we analyzed the expression of a wide range of epithelium-derived cytokines in normal mammary glands, premalignant lesions, and late-stage tumors of MMTV-PyMT (PyMTtg) and K14-Cre, Brca1f/f, p53f/f mouse models. These mice spontaneously develop breast tumors that resemble human luminal and basal-like breast cancers, respectively. Of the cytokines examined, IL-1a was highly expressed in premalignant lesions compared to mammary glands and tumors. Additionally, IL-12p40 was upregulated in both early and late stages of breast cancer compared to normal mammary gland. Other cytokines, like IL-7, IL-11, and IL-24, were highly expressed in the mammary gland, but their expression was lost during breast tumorigenesis. In conclusion, we identified upregulation of IL-1a and IL-12p40 in early-stage breast cancer, suggesting a role for these cytokines in inducing antitumor immunity in the breast. Furthermore, we demonstrate selection against expression of IL-7, IL-11, and IL-24 during the process of cancer initiation and promotion, suggesting a role in normal mammary gland homeostasis and possibly immunity against breast carcinogenesis. Taken together, we have identified potential novel therapeutic targets that can be leveraged for breast cancer immunoprevention.

Citation Format: Sanne Kroon, Kaitlin Dempsey, Margherita Boieri, Shawn Demehri. Epithelium-derived cytokines in breast carcinogenesis [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B102.
Abstract B12: Exploiting the zebrafish to study lymphocyte infiltration in MYCN-driven neuroblastoma
Authors: Qin Xiaodan , Lam Andrew , Zhang Xike , Hurlstone Adam , Feng Hui
Published: March 2020

Application of tumor immunotherapy has achieved an unprecedented success in patients with hematopoietic malignancies. However, it remains challenging to treat the majority of solid tumors with immunotherapy due to low lymphocyte infiltration, treatment resistance, and immunosuppressive tumor microenvironment. Improving the clinical efficacy of immunotherapy in solid tumors requires a deeper understanding of immune evasion. Neuroblastoma is the most common extracranial tumor in children and is responsible for ~15% of childhood cancer-related deaths. MYCN is a member of the MYC oncogene family, and its aberrant expression in neuroblastoma predicts aggressive disease and poor prognosis. This largely results from the low infiltration rates of cytotoxic lymphocytes in MYCN-amplified neuroblastoma. Elucidation of the mechanism by which MYCN suppresses antitumor response in the neuroblastoma microenvironment will provide useful information to guide immunotherapy. Here we generated a compound transgenic zebrafish, Tg(dβh:MYCN;dβh:EGFP;lck:EGFP;CD4:mCherry), in which lymphocytes and tumor cells are differentially fluorescently labeled. We first monitored the infiltration of mCherry-labeled CD4+ T cells in EGFP-positive MYCN-overexpressing premalignant neural crests at 7, 14, and 21 days post fertilization (dpf). We found that at day 14 and 21, MYCN-overexpressing premalignant neural crests can attract a significantly higher number of lymphocytes, compared to control neural crests without MYCN overexpression. Interestingly, we also found the increased number of infiltrated T cells was MYCN dose dependent by using low MYCN-expressing transgenic fish, Tg(dβh:MYCN;dβh:mCherry;lck:EGFP). To determine if T-cell development is impacted, we imaged the thymus of MYCN-overexpressing or control fish at day 7, 14, and 21 dpf. We found that fluorescent intensity of thymus is significantly higher in MYCN-overexpressing fish compared to control fish. Taken together, our study identified MYCN-overexpressing premalignant neural crests can not only affect T-cell development but also attract T cells to disease site. We will perform further experiments to understand how and why MYCN-overexpressing premalignant neural crests impact T-cell development and which population of T cells is attracted to disease site.

Citation Format: Xiaodan Qin, Andrew Lam, Xike Zhang, Adam Hurlstone, Hui Feng. Exploiting the zebrafish to study lymphocyte infiltration in MYCN-driven neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B12.
Abstract B19: Cowpea mosaic virus stimulates antitumor immunity through recognition by MYD88-dependent signaling of multiple Toll-like receptors
Authors: Mao Chenkai , Beiss Veronique , Shukla Sourabh , Steinmetz Nicole , Fiering Steven
Published: March 2020

Cowpea mosaic virus (CPMV), a non-enveloped plant virus, and empty CPMV (eCPMV), a virus-like particle composed of CPMV capsid, have demonstrated strong activation of neutrophils, suppression of primary tumor growth, and durable systemic antitumor immunity in murine models with no side effects when administered intratumorally as an in situ vaccine. However, it is not known how immune cells recognize these nanoparticles. In this study, CPMV and eCPMV exhibited overlapping yet distinctive recognition patterns and immunostimulatory profiles. Specifically, CPMV generated stronger selective induction of cytokines and chemokines in naive splenocytes and exhibited more potent antitumor efficacy compared to eCPMV. We found that MyD88 is required for both CPMV- and eCPMV-elicited immune responses. Screening with HEK-BlueTM mouse and human Toll-like receptor (TLR) assays along with response and efficacy studies in corresponding TLR-/- mice indicated the recognition of both CPMV and eCPMV by MyD88-dependent TLR2 and TLR4 as well as recognition of CPMV by TLR7. Furthermore, the studies revealed that TLR2 and TLR4 are complementary to each other in recognizing the capsid of either particle in vivo and in vitro. We further confirmed that secretion of type I interferons (IFNs) by CPMV is critical to its stronger efficacy, which is dependent on the binding between TLR7 and the encapsulated single-stranded RNAs (ssRNAs) of CPMV. These findings link efficacy with molecular recognition and provide rationale for developing more potent viral particles as immunotherapeutic vaccines to cancer.

Citation Format: Chenkai Mao, Veronique Beiss, Sourabh Shukla, Nicole Steinmetz, Steven Fiering. Cowpea mosaic virus stimulates antitumor immunity through recognition by MYD88-dependent signaling of multiple Toll-like receptors [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B19.