bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1286: Oral administration of poly gamma glutamic acid significantly enhances the antitumor immune response of doxorubicin in a murine cancer model through regulating tumor microenvironment
Authors: Choi Joungwoo , Jung Solmin , Oh Yena , Jang Jaepyeong , Cho Yeondong , Lee Do young , Park Youngcheol , Jeong Kwangil
Published: June 2022

Immunogenic cell death (ICD) is a type of cancer cell death triggered by certain chemotherapeutic drugs, oncolytic viruses, and other therapies. In particular, doxorubicin (DOX), one of the chemo-drugs, is well known to induce ICD, which involves the activation of the immune system against cancer in immunocompetent hosts. ICD comprises the release of damage-associated molecular patterns (DAMPs) from dying tumor cells that result in the activation of tumor-specific immune responses, thus eliciting long-term efficacy of anticancer drugs by combining direct cancer cell killing and antitumor immunity. However, the antitumor immunity can be achieved by overcoming the immunosuppressive tumor microenvironment (TME). Most current treatment strategies harnessing the tumor microenvironment focus on T cell-immune response, either by promoting activating signals or suppressing inhibitory ones.We here present a novel approach to cope with the inhibitory TME, combinatory treatment of DOX with poly gamma glutamic acid (γ-PGA). PGA, a safe edible biomaterial secreted by B. subtillis, has been evaluated in the clinical trials with the patients diagnosed as cervical intraepithelial neoplasia 1 (CIN 1). From the study using murine cancer models, including MC38, we found the combinatory treatment with γ-PGA restored the downregulation of NK cell activity caused by DOX, enhanced ICD and T cell responses, and reduced proinflammatory cytokines in the tumor tissues. Taken together, co-administration of γ-PGA exhibited synergistic anti-cancer effects by favoring the immunosuppressive TME in the combinatory treatment with conventional chemo-drugs.

Citation Format: Joungwoo Choi, Solmin Jung, Yena Oh, Jaepyeong Jang, Yeondong Cho, Do young Lee, Youngcheol Park, Kwangil Jeong. Oral administration of poly gamma glutamic acid significantly enhances the antitumor immune response of doxorubicin in a murine cancer model through regulating tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1286.
Abstract 1338: Humanized mouse model for in vivo evaluation of bispecific antibody efficacy
Authors: Yao Li-Chin , Keck James G. , cai Danying
Published: June 2022

Bispecific antibodies have shown several promising advantages over monoclonal antibodies and are emerging as one of growing cancer immunotherapies. Preclinical in vivo models to assess efficacy associated with immune cell engagement are needed. Here we evaluated an EGFR bispecific T cell engager (EGFRXCD3 BiTE) using xenograft models in humanized NSG™ (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice that have been engrafted with human CD34+ HSC and have human T cells in circulation. Three EGFR+ tumor models were used in this report including LG1202 lung PDX and two triple negative breast cancer cell lines MDA-MB-231 with a high EGFR expression level and MDA-MB-453 with a low EGFR expression. Interestingly we found that all three models were resistant to EGFR monoclonal antibody drug cetuximab despite the expression of EGFR but responded to EGFRXCD3 BiTE. BiTE-induced T cell activation was evident through the analysis of tumor infiltrated lymphocytes by flow cytometry at the end of the study. BiTE treatment for 3 weeks effectively reduced the LG1202 tumor growth whereas the effect of cetuximab on the tumor growth was minimal. In the BiTE-treated group, CD3+ T cell percentage increased as compared to the vehicle group, and both CD4+ and CD8+ T cell subsets showed an increased frequency of CD69+ cells. We also observed elevated levels of human cytokines IFN-γ, IL-6, IL-2, and IL-10 in circulation 4 h after animals received the first BiTE dose. We evaluated the dose response of BiTE treatments on MDA-MB-231 model with a high EGFR expression. Low dose BiTE reduced tumor growth whereas high dose BiTE effectively prevented or even regressed some tumor growth. Flow analysis showed that tumor CD3+ T cell frequency remained similar to the vehicle group but CD8+ T frequency increased after high dose BiTE. High CD69+CD8+ T cell frequency was found in the vehicle group and the percentage further increased after high dose BiTE. For the low EGFR-expressing MDA-MB-453 model, BiTE treatment for 4 weeks also effectively reduced the tumor growth. In comparison, while cetuximab treatment decreased EGFR expression on tumor cells, it did not reduce the tumor growth. Flow analysis showed that BiTE-treated group had increased T cell infiltration into the tumors when compared to the PBS and cetuximab groups. The frequency and cell number of CD25+ and CD69+ CD4+ T and CD8+ T cells in the tumor increased after BiTE treatment. Together, these data support our humanized mouse model to be used to validate T-cell dependent bispecific antibody efficacy.

Citation Format: Li-Chin Yao, James G. Keck, Danying cai. Humanized mouse model for in vivo evaluation of bispecific antibody efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1338.
Abstract 135: Age-related immunosenescence and cancer drug toxicity and efficacy in C57BL/6J syngeneic model
Authors: Yang Jiwon , Schile Andrew , Cheng Mingshan , Keck James G.
Published: June 2022

Cancer has a higher incidence in the elderly and immunotherapy is widely applied to this population. However, the majority of preclinical studies assessing drug toxicity and efficacy have been done in young healthy mice. Immunosenescence is a state of aging-related gradual deterioration of the immune system in the elderly. Here, we established tumor models in aged mice that show immunosenescence for a better representation of cancer patients, and to examine the effects of aging and immunosenescence on cancer drug efficacy and toxicity. Young (7 weeks), mid-aged (54-58 weeks), and aged (76-78 weeks) C57BL/6J female mice were used for this study. Syngeneic tumor models were established using three different mouse cancer cell lines, B16-F10 melanoma, MC38 colon adenocarcinoma, and E0771 breast cancer. Age-related tumor growth kinetics varied based on tumor models. Specifically, aging did not affect tumor growth kinetics of B16-F10 tumors, while aged mice showed slowed growth of MC38 tumors and faster growth of E0771 tumors. To evaluate the maximum tolerated dose, young and aged mice were dosed with the standard of care cancer drugs (docetaxel, cisplatin, and doxorubicin) and body weight was monitored three times a week. Aged mice showed higher toxicity against standard care cancer drugs compared to young counterparts. Next, we performed efficacy study using checkpoint inhibitor. Young, mid-aged, and aged mice bearing MC-38 tumor were treated with isotype control or Anti-PD1 (8 mg/kg), intravenously, twice weekly for 3 weeks. Whole blood and tumor samples were collected for immune cell profiling. Comparisons of PD-1 response in the various age groups showed that anti-PD1 treatment showed significant suppression of tumor growth in all age groups with the MC38 tumors, with the greatest effect in aged mice. We also found that anti-PD1 treatment increased T cell infiltration into the tumor, and that regulatory T cells in the tumor are decreased in aged mice. In conclusion, we established syngeneic tumor models using aged mice, and found aged mice exhibit higher toxicity against drugs. The effect of aging on tumor growth kinetics varies depending on the models. Differences in immune cell infiltration in the tumor might be an underlying event that causes age-related differences in response to anti-PD1 treatment. The integration of aging in cancer research could potentially bridge the gap between preclinical studies and clinical outcomes.

Citation Format: Jiwon Yang, Andrew Schile, Mingshan Cheng, James G. Keck. Age-related immunosenescence and cancer drug toxicity and efficacy in C57BL/6J syngeneic model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 135.
Abstract 1363: Preclinical development of bispecific CAR-T cells for canine B cell malignancies
Authors: Atherton Matthew J. , Rotolo Antonia , Mason Nicola J.
Published: June 2022

Chimeric antigen receptor (CAR)-T cells targeting CD19 have revolutionized the treatment of B cell malignancies in people. However, post-treatment loss of CD19 has emerged as an adaptive mechanism of resistance. Pet dogs that develop tumors spontaneously in an immune competent setting represent a compelling parallel population of patients to study next generation cancer immunotherapies. Comparative trials at the University of Pennsylvania are investigating the activity of canine CAR-T cells targeting either CD19 or CD20 in canine B cell lymphoma patients and have revealed a favorable safety profile with early signs of anti-tumor activity. Aligned with experiences in human trials, expansion of CD20-negative B cells has been observed in dogs treated with anti-CD20 CAR-T cells. Bispecific CAR-T cells that simultaneously target two antigens to mitigate antigen escape have encouraging efficacy profiles in human patients. Thus, we designed a dual-targeting CAR specific for canine B cell antigens. A second-generation canine CAR construct targeting both CD19 and CD20 with a 28z signaling domain was cloned into the MSGV1 backbone. Chimeric retrovirus encoding RD114 and VSV-G envelope proteins and the CAR construct was made using GP2-293 cells. Jurkat NFAT-GFP reporter cells and primary T cells from dogs diagnosed with high grade B cell lymphoma were retrovirally transduced with bispecific CARs. Antigen specific reactivity was assessed against cell lines with variable CD19 and CD20 expression patterns. The CD19-CD20 CAR was efficiently transduced, with stable expression in both Jurkat and primary canine T cells. Jurkat reporters revealed dual-specific CAR signaling after co-culture with CD19+ and/or CD20+ target cells. Primary canine CAR-T cells expanded in vitro and exhibited CAR-specific proliferation and cytotoxicity against CD19+ CD20+ target cells. Preclinical manufacture of functional, bispecific anti-CD19 anti-CD20 canine CAR-T cells is feasible. A clinical trial recruiting dogs diagnosed with B cell cancer will assess safety and efficacy of this approach in vivo, with the aim of establishing the dog as a compelling model to appraise the ability of bispecific CAR-T cells to overcome therapeutic failures secondary to antigen loss.

Citation Format: Matthew J. Atherton, Antonia Rotolo, Nicola J. Mason. Preclinical development of bispecific CAR-T cells for canine B cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1363.
Abstract 1393: A Novel platform providing automated, consistent and gentle cell isolation from tissues for downstream applications
Authors: Yu Liping
Published: June 2022

Tumor biologists have long studied tumor tissue to understand cancer progression and to ultimately treat the disease. The gold standard would be to analyze tumor and immune cells from the same sample, providing purified cells that are interacting within the tumor. Many tissue homogenization techniques have been developed to release cells for downstream analysis, however, an automated and gentle process of purifying multiple cell types from the same processed tissue has remained elusive. The industry-standard workflows, such as centrifugation, have presented long-standing performance issues in terms of low cell recovery and inefficient removal of molecular debris, which often leads to sequencing results that are difficult to interpret. To ensure the quality of sequencing data from tissues, upstream processes need to improve to get the most from the precious patient tissue. Applied Cells has developed an integrated system based on multiple physics principles to achieve cell wash, concentration, and target cell isolation, with high recovery at an unprecedented flow rate. This platform combines tunable, acoustic cell purification and in-flow, column-free immuno-magnetic separation technologies, enabling automation of the entire cell sample preparation workflow for genomics and transcriptomics analysis. By harnessing the power and precision of acoustic cell manipulation, the MARS System can process a variety of samples including dissociated solid tissue in enzymatic buffer with high cell recovery and superior molecular debris removal compared to standard methods. For instance, when purifying leukocytes from a lung cancer biopsy tissue samples, MARS can remove 99.50% debris and 91% of dead cells in one single step, which is supreme than the conventional centrifuge cell wash while having >90% leukocyte recovery at 1mL/min flow rate. The in-flow microfluidics design drastically reduces residual DNA and RNA fragments that are difficult to remove using centrifuge wash methods. Furthermore, the system enables parallel cell purification and concentration for high throughput sample processing. Tumor or TIL cells can be further enriched by means of positive magnetic selection for genomic or cell biology applications. This novel platform allows improved tumor and TIL recovery in a gentle, consistent manner while having minimal interference with downstream applications, all from a single source starting material.

Citation Format: Liping Yu. A Novel platform providing automated, consistent and gentle cell isolation from tissues for downstream applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1393.
Abstract 1439: Meta-analysis of the NQO1 gene C609T polymorphism: Increased risk of colorectal cancer
Authors: Ganji Chaithanya , Basha Raasil R. , Bhaskar Lakkakula V.K.S. , El-Rayes Bassel F.
Published: June 2022

Background/objective: Colorectal cancer (CRC) is aggressive and widespread (3rd among malignancies) in the United States. Recent studies suggest that metabolic pathways play an essential role in CRC. NQO1 is an enzyme that contributes to chemoprotection for CRC. Previous studies showed that the link between the NQO1 polymorphisms and CRC risk yielded conflicting results. The aim of this meta-analysis is to test the connection between NQO1 C609T polymorphisms and CRC risk.

Methods: PubMed, Web of Science, and Google Scholar were used in bibliographic searches until July 17, 2021. For the NQO1 C609T analysis, a total of 18 studies (7568 CRC cases and 9003 controls) were considered. Data was gathered and subsequently calculated using the pooled odds ratio (OR) (95% confidence interval, CI).

Results: The relation between NQO1 polymorphism and CRC risk (T vs. C: OR=1.17, 95% CI=1.07-1.29, p<0.001; TT + CT vs. CC: OR=1.19, 95% CI=1.06-1.34, p<0.001) was significant. Furthermore, stratified investigation based on ethnicity indicated significant association between NQO1 polymorphism and CRC risk in only Caucasian populations (T vs. C: OR=1.15, 95% CI=1.08-1.24, p<0.001; TT + CT vs. CC: OR=1.17, 95% CI=1.08-1.27, p<0.001). No proof of bias in publications was found for NQO1 polymorphism (Eggers p=0.929).

Conclusion: This study indicates that the C609T polymorphism of NQO1 is linked with CRC risk in Caucasian populations.

Citation Format: Chaithanya Ganji, Raasil R. Basha, Lakkakula V.K.S. Bhaskar, Bassel F. El-Rayes. Meta-analysis of the NQO1 gene C609T polymorphism: Increased risk of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1439.
Abstract 1445: Influence of neighborhood deprivation on the DNA epigenome in cancerous breast tissue from African American and European American women
Authors: Lord Brittany D. , Rossi Emily , Dorsey Tiffany , Kiely Maeve , Hutchinson Ruby , Ambs Stefan
Published: June 2022

Low socioeconomic status (SES) associates with early onset of chronic diseases and reduced life-expectancy. This relationship is only partly explained by unhealthy behavioral habits and inadequate access to health care. Chronic life stress is more prevalent in low SES communities and has been shown to affect DNA methylation and the immune system. Yet, the biological processes that mediate the impact of SES on health to promote the development of chronic diseases like cancer remain poorly understood. Our study aims to uncover whether DNA methylation is linked to neighborhood socioeconomic deprivation and to a biology that causes inflammation and changes to the immune environment, thereby promoting breast cancer progression and affecting response to therapy. These differences have been characterized in 181 breast tumors (108 African American (AA), 73 European American (EA)). We geocoded the addresses of our participants and linked these data to a Census-level neighborhood deprivation index (NDI). Statistically significant associations between NDI, race and methylation beta values were determined using linear regression followed by Benjamini-Hochbergcorrection. Of the 18 CpG sites that differed between individuals with low and high neighborhood deprivation, 3 were hypermethylated in women with high neighborhood deprivation. We also used methylCIBERSORT to estimate immune subpopulation differences by race group, breast cancer subtype, and ND. For AA women with aggressive triple negative breast cancer, neighborhood deprivation was significantly positively correlated with absolute immune cell scores (p=0.011), including a higher absolute score for endothelial cell expression (p=0.006) and eosinophils (p=0.049). Our findings give insight into how socioeconomic position and neighborhood deprivation may affect cancerous mammary gland biology by altering DNA methylation patterns.

Citation Format: Brittany D. Lord, Emily Rossi, Tiffany Dorsey, Maeve Kiely, Ruby Hutchinson, Stefan Ambs. Influence of neighborhood deprivation on the DNA epigenome in cancerous breast tissue from African American and European American women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1445.
Abstract 1547: LncRNA UCA1 modulates glucose metabolism proteins in colorectal cancer
Authors: Leslie Sophia , Doxtater Kyle , Lopez Samantha , Anilkumar Adithya , Jaggi Meena , Chauhan Subhash , Tripathi Manish
Published: June 2022

Colorectal cancer (CRC) is the primary cause of cancer-related deaths worldwide. According to the American Cancer Society, it is the third most common cancer diagnosed in adults in the United States. It will have an estimated 149,500 new cases of colon cancer in 2021. In addition, studies show CRC has increased morbidity with obesity and obesity-related diseases such as diabetes. In a geographical area of The Rio Grande Valley which has a high incidence of diabetes, understanding the connection between CRC and diabetes is essential. Our lab identified LncRNA UCA1, a poor prognosis marker in CRC, responsible for increased proliferation and upregulating glucose metabolic pathway. First, we investigated the possible role of UCA1 in glucose metabolism, finding that UCA1 overexpressing cells (SW480+UCA1) showed a higher glucose consumption than their vector. The inverse of this trend was established when we knockdown UCA1 (SW620+shUCA1). Further, we found that overexpressing UCA1 increases the migration, invasion, and proliferation of SW480 cells. A vital aspect of the metastatic progression is when the cell survives detachment from the extracellular matrix. A route in which they overcome apoptosis is through modulation of the metabolic pathways, upregulating such markers as GLUT1, FDFT1, SGK1, and HIF1α. At 36hrs of anchorage-independent stimulation, we found an uptick in glucose consumption and lactate production and an increase in the expression of GLUT1, FDFT1, SGK1, and HIF1α compared to 0hrs. The Warburg effect is when glycolysis is no longer connected to the tricarboxylic acid cycle or oxidative phosphorylation. The increase in expression of the markers GLUT1, FDFT1, SGK1, and HIF1α indicates aerobic metabolic activity during anchorage-independent growth with UCA1 expression. This relationship needs to be further examined and could lead to understanding the increased morbidity associated with diabetes and CRC.

Citation Format: Sophia Leslie, Kyle Doxtater, Samantha Lopez, Adithya Anilkumar, Meena Jaggi, Subhash Chauhan, Manish Tripathi. LncRNA UCA1 modulates glucose metabolism proteins in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1547.
Abstract 1559: Repurposing of pimavanserin tartrate for the treatment of glioblastoma
Authors: Zabel Carson , Ramachandran Sharavan , Srivastava Sanjay
Published: June 2022

Glioblastoma is a highly aggressive brain tumor that has a median survival of 15 months. We aim to give a substitute to the current chemotherapeutic drug temozolomide by using the FDA-approved anti-psychotic pimavanserin tartrate (PVT). To uncover the effectiveness of PVT, we did cytotoxicity assays using SRB to determine the percentage of cell survival in a dose-dependent and time-dependent manner. The apoptotic effects were measured using Annexin-V/APC assay. The mechanism of action was determined utilizing Western Blotting. In different glioblastoma tumor cell lines, the IC50 for 24 hours was between 6 µM and 10 µM. 48 and 72 hours had an IC50 between 4 µM and 6 µM. SF188 glioblastoma cell line had approximately 70% to 80% more apoptotic cells in 10 µM PVT when compared to control in Annexin-V/APC assay. PVT causes a strong modulation of the PI3K/AKT and MAPK pathways when given in increasing concentration at 48 hours. The level of apoptosis was confirmed through Western Blot as well using apoptotic markers. The preliminary data show the ability of PVT to cause apoptosis of glioblastoma tumor cells in vitro through the AKT and MAPK signaling.

Citation Format: Carson Zabel, Sharavan Ramachandran, Sanjay Srivastava. Repurposing of pimavanserin tartrate for the treatment of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1559.
Abstract 1609: Oxidative stress triggers tumor edge progression of tumor microenvironment in the minipig spinal cord glioma model
Authors: Lei Kecheng , Tora Muhibullah S. , Neill Stewart G. , Nagarajan Purva P. , Federici Thais , Canoll Peter , Boulis Nicholas M.
Published: June 2022

Oxidative stress is exerted by reactive oxygen species (ROS) that accumulate due to an imbalance between ROS generation and elimination, which impacts cellular metabolism and consequently tumorigenesis in the tumor microenvironment. Recently, our group has developed a spinal cord glioma (SCG) model in Göttingen minipigs. However, the mechanism of how SCGs balance redox and thereby modulate cellular proliferation is unclear. Here, we demonstrated that the NRF2/NQO1 signaling, known to mediate oxidative stress, is upregulated in unresectable SCG cells infiltrating at the leading edge compared with the core cells. Moreover, pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, were significantly elevated in the edge SCG cells. Immunohistochemistry demonstrated positive staining for a higher Ki-67 proliferative index, GFAP, SOX2 and Olig2 in the edge cells compared to the core cells. The oxidative metabolic heterogeneity of immune and stromal cell subtypes among tumor samples was also explored. Hence, our study demonstrated the tumor microenvironment and involvement of the NRF2/NQO1 pathway for redox homeostasis in our minipig SCG model, which can be used to explore targets of the pre-clinical treatment investigations in SCG.

Citation Format: Kecheng Lei, Muhibullah S. Tora, Stewart G. Neill, Purva P. Nagarajan, Thais Federici, Peter Canoll, Nicholas M. Boulis. Oxidative stress triggers tumor edge progression of tumor microenvironment in the minipig spinal cord glioma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1609.
Abstract 1643: B-hIL12A/hIL12B/hIL12RB1/hIL12RB2: A novel animal model for generation of IL12 therapies
Authors: Wang Linlin , Liu Chang , Chromikova Veronika , Lyu Ruili , Jin Tianquan , Kuraguchi Mari
Published: June 2022

The pro-inflammatory cytokine, interleukin 12 (IL12), is primarily responsible for cell-mediated immunity and is chiefly produced by antigen presenting cells. Prior preclinical studies elucidated a promising role for IL12 mediating anti-tumor effects, however, clinical studies using cancer patients showed disappointing efficacy results, as well as severe adverse effects, with administration of systemic IL12 therapies. Intriguingly, progress in targeted delivery to the tumor microenvironment renewed high expectations on IL12 as a promising anti-tumor drug either alone or in combination due to its ability to inhibit and/or reprogram immunosuppressive cell subsets. Therefore, to evaluate the efficacy of IL12 and IL12 analogs in preclinical studies, Biocytogen has successfully generated a novel hIL12A/hIL12B/hIL12RB1/hIL12RB2 knock-in mouse strain. In this mouse model, the full coding sequences (CDS) of the mouse Il12a or Il12b gene were replaced by their human counterpart IL12A or IL12B gene sequences respectively. In addition, chimeric CDS, composed of human IL12RB1 or IL12RB2 extracellular region plus mouse Il12rb1 or Il12rb2 cytoplasmic region, was inserted into the mouse endogenous gene locus in in these animals, to express an IL12 receptor (IL12R) capable of recognizing human IL12 signaling. Expression analysis by RT-PCR detected human IL12A/IL12B/IL12RB1/IL12RB2 mRNA in humanized mice but not in wild-type mice. Additionally, human IL12 p70 was successfully detected in the serum of humanized mice by ELISA. The immune cell subpopulations in spleen, blood, and lymph node did not differ between humanized mice and wild-type mice suggesting no impact on cell differentiation and maturation. Anti-tumor activity of a modified human IL12 was assessed in humanized mice bearing colon cancer MC38 cells and showed tumor inhibitory effects. Moreover, the tumor growth recovered when the administration of the drug was stopped. Altogether, B-hIL12A/hIL12B/hIL12RB1/hIL12RB2 humanized mice provide a promising preclinical efficacy model for human IL12 therapy alone or in combination. Keywords: IL12, humanized mice, antibody efficacy

Citation Format: Linlin Wang, Chang Liu, Veronika Chromikova, Ruili Lyu, Tianquan Jin, Mari Kuraguchi. B-hIL12A/hIL12B/hIL12RB1/hIL12RB2: A novel animal model for generation of IL12 therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1643.
Abstract 1645: Advancing the development of CD16A-based bispecific antibodies in humanized CD16A mouse models
Authors: Chang Jiahui , Li Tao , Ma Limei , Sun Xiaoxia , Shen Yuelei
Published: June 2022

Antibody-dependent cell cytotoxicity (ADCC) is an important mechanism in immunotherapy. CD16A (FcγRIIIA), a low-affinity receptor for the IgG Fc domain, facilitates ADCC and is responsible for triggering the lysis of target cells by natural killer (NK) cells. Human CD16A is closely related to mouse Fcgr4 and is expressed in NK cells and macrophages, but is absent in granulocytes. Alternatively, mouse Fcgr4 is expressed in granulocytes and macrophages and is absent in NK cells. CD16A-based bispecific antibodies targeting NK cells have been studied for cancer therapy. To evaluate the anti-tumor efficacy of CD16A-based bispecific antibody in mouse tumor models, we have successfully generated a novel humanized CD16A knock-in mouse strain (B-hCD16A Mice). Considering the differential expression patterns between human CD16A and mouse Fcgr4, the regulatory region and whole genome sequence of mouse Fcgr4 were replaced by the human CD16A gene. Human CD16A and mouse Fcgr4 protein expression was confirmed in heterozygous B-hCD16A mice by FACS and RT-PCR. Human CD16A protein was detected in NK cells and macrophages of B-hCD16A mice, but not granulocytes. When compared with wild type mice, there were no abnormalities in the populations of immune cells in B-hCD16A mice. CD16A-based bispecific antibody usually binds to CD16A on NK cells, whereas the other arm of the antibody binds to tumor antigen on cancer cells. Initial assessment of the tumorigenicity of MC38 cells were evaluated in B-hCD16A mice, and results indicated that these cancer cells were able to establish tumors in vivo. These data suggest that the B-hCD16A mouse model is an effective tool for future in vivo efficacy evaluation of therapeutic CD16A-based bispecific antibodies prior to clinical development.

Citation Format: Jiahui Chang, Tao Li, Limei Ma, Xiaoxia Sun, Yuelei Shen. Advancing the development of CD16A-based bispecific antibodies in humanized CD16A mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1645.
Abstract 1649: Humanized PVRIG mice: A novel tool for evaluating anti-PVRIG immunotherapies
Authors: Zhang Yifu , Yao Jiawei , Soto Rebecca , Liu Chonghui , Lin Qingcong
Published: June 2022

The use of humanized mouse models provides a promising platform for preclinical studies aimed at developing novel immunotherapies. While current checkpoint inhibitors have improved patient outcomes, combination therapy targeting additional checkpoint receptors may heighten the immune response. Specifically, previous reports have shown that high expression of the immune checkpoint poliovirus receptor-related immunoglobulin domain-containing (PVRIG) protein leads to T-cell exhaustion. Furthermore, in vitro blockade of human PVRIG enabled T-cell activation. To explore the translational potential of inhibiting PVRIG, we evaluated the efficacy of PVRIG antibodies by generating a humanized B-hPVRIG mouse model. In this model, the mouse Pvrig gene was replaced by the human counterpart, and expression of the human PVRIG protein was confirmed in homozygous B-hPVRIG mice. Additionally, flow cytometry analysis showed similar distribution of T-cell subtypes within the blood, spleen, and lymph nodes of B-hPVRIG mice compared to wild-type C57BL/6. Lastly, our in vivo efficacy studies showed that homozygous B-hPVRIG mice that were subcutaneously implanted with murine colon cancer (MC38) cells and treated with 30 mg/kg of the human PVRIG antibody, reached a tumor growth inhibition (TGI) of approximately 40%. This data suggests that human PVRIG antibodies significantly inhibit tumor growth in homozygous B-hPVRIG mice. Altogether, our humanized B-hPVRIG mouse model is an effective tool for in vivo efficacy studies aimed at the development of novel PVRIG immunotherapies.

Citation Format: Yifu Zhang, Jiawei Yao, Rebecca Soto, Chonghui Liu, Qingcong Lin. Humanized PVRIG mice: A novel tool for evaluating anti-PVRIG immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1649.
Abstract 1651: Generation of humanized TLR8 mice for the evaluation of human TLR8 agonists
Authors: Shen Yuelei , Guo Yanan , Kong Lingqi , Bai Yang Bai , Yao Jiawei , Shang Chengzhang , Huang Jingwen
Published: June 2022

Toll-like receptor 8 (TLR8) is encoded by the TLR8 gene and belongs to the family of toll-like receptors (TLRs). TLR8 is predominantly expressed in lung and peripheral blood leukocytes. Close to the TLR8 gene locus, another TLR family member, TLR7, is also located on chromosome X and potentially can be manipulated by TLR8. TLR8 is an endosomal receptor that recognizes single-stranded RNA (ssRNA) viruses. After exogenous ssRNA infection, TLR8 will recruit MyD88 and activate downstream signaling. Similarly, miRNAs secreted from tumor cells also can activate TLR8 and lead to activation of the transcription factor NF-κB. Therefore, TLR8 also has been recognized as a potential therapeutic target. TLR8 agonists (e.g. VTX-2337) are undergoing clinical trials as immune stimulants in combination therapy for some cancers. Immune-stimulating antibody conjugates (ISACs) were developed by Bolt therapeutics, comprising a TLR7/8 dual agonist conjugated to tumor-targeting antibodies. ISACs can drive tumor killing by TLR mediated activation of myeloid cells and subsequent T-cell-mediated antitumor immunity, resulting in tumor clearance and immunological memory. Although human TLR8 arises as a promising target, emerging therapeutic candidates are lacking suitable mouse models on the market for preclinical in vivo screening and efficacy evaluation. Biocytogen has developed a novel TLR8 humanized mouse model (B-hTLR8 mice). Exon 3 of the mouse TLR8 gene, which encodes the extracellular domain, was replaced by human TLR8 counterparts. Human TLR8 protein and mRNA were detected in lung tissue from homozygous B-hTLR8 mice with the absence of mouse TLR8. Additionally, human TLR8 was detectable via flow cytometry in the dendritic cells and monocytes. To further validate the model, we have examined the model in vivo with a benchmark TLR8 agonist GS-9688. We observed TNFα secretion in B-hTLR8 mice but not in wild-type mice. Therefore, B-hTLR8 mouse model is a promising model for preclinical in vivo studies to evaluate TLR8 agonists and antibody-conjugated TLR8 agonists.

Citation Format: Yuelei Shen, Yanan Guo, Lingqi Kong, Yang Bai Bai, Jiawei Yao, Chengzhang Shang, Jingwen Huang. Generation of humanized TLR8 mice for the evaluation of human TLR8 agonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1651.
Abstract 1652: A humanized mouse model of the promising immune checkpoint molecule B7-H4
Authors: Lyu Ruili , Liu Chonghui , Charpentier John , Zhao Suman , Yu Luke (Zhaoxue)
Published: June 2022

B7-H4, a member of the B7-family of immune-regulatory ligands, has emerged in recent years as a promising target for immune checkpoint therapy development. B7-family ligands bind via extracellular tandem immunoglobulin-like domains to CD28-family receptors on lymphocytes to communicate co-stimulatory or co-inhibitory signals. B7-H4 remains an orphan ligand, although evidence from knockout mice suggests a co-inhibitory role and its expression on cancer cells and tumor-associated macrophages is associated with inhibition of proliferation, cell cycle progression, and cytokine secretion in T cells. B7-H4 is highly expressed in a variety of tumors (e.g., renal, gastric, breast, and ovarian carcinomas) and plays less well-appreciated roles in autoimmune diseases, viral infections, and maternal-fetal immunity. Negative regulation of T cell responses by B7-H4 has also been shown to induce tolerance of islet allografts in mouse models of Type I diabetes. A range of B7-H4-targeted immunotherapy agents have been successfully developed, including monoclonal antibodies, antibody-drug conjugates, B7-H4/CD3-targeting bispecific antibodies, and B7-H4-specific CAR-T cells. To evaluate the efficacy of a B7-H4 antibody, Biocytogen generated humanized B-hB7-H4 mice. Our targeting strategy was to replace the murine B7-H4 extracellular domain sequences with their corresponding human sequences. We verified mRNA and protein expression of human B7-H4 in the humanized B-hB7-H4 mice by RT-PCR and Western Blot, respectively. An in vivo efficacy study demonstrated that B7-H4 antibodies significantly inhibited tumor growth in B-hB7-H4 mice bearing tumors derived from the B16-F10 murine melanoma line. These data confirm that the B-hB7-H4 humanized mouse model is a powerful tool for evaluating the preclinical potential of B7-H4-targeting immune-therapeutics. Keywords: Humanized mice, B7-H4, Immunotherapy

Citation Format: Ruili Lyu, Chonghui Liu, John Charpentier, Suman Zhao, Luke (Zhaoxue) Yu. A humanized mouse model of the promising immune checkpoint molecule B7-H4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1652.
Abstract 1673: Establishment and characterization of pediatric brain tumor models in an orthotopic mouse model
Authors: Oswald Eva , Lashuk Kanstantsin , Gojo Johannes , Lenhard Dorothee , Mack Norman , Krausert Sonja , Lötsch Daniela , Jones David , Kool Marcel , Milde Till , Berger Walter , Pfister Stefan M. , Schüler Julia
Published: June 2022

Brain and spinal cord tumors are the second most common group of cancers in children. For children who experience relapses of their tumors, usually after very intensive first-line therapy, curative treatment options are scarce. Thus, the need for predictive preclinical platforms explicitly for pediatric brain tumors is an urgent need. In the framework of the ITCC-P4 public-private partnership, we thus far fully established 23 pediatric brain tumor-derived PDX models. A selection of four high grade glioma, one medulloblastoma and one ependymoma models were used to validate a protocol for fluorescence-based optical imaging. Tumor growth characteristics, latency and histopathology were evaluated for the un-transduced and iRFP713-transduced models side by side. In case of iRFP713-transduced PDX models, tumor load was determined twice a week with the Pearl trilogy system (LiCor, Germany). All animals were examined for neurological symptoms daily and body weight was examined twice a week. The latency of the tumor models ranged from 29 days to 180 days. The take rate was 100% across all the models with n=12 NSG mice per setting. The transduction did not influence the take rate, but 30 - 40% more donor material was needed due to viability loss during the overnight transduction. It was not possible to determine the transduction efficiency for iRFP713 in the overnight culture as the signal is getting upregulated only 48h - 72h post transduction, e.g., when the cells are already implanted. The signal was stable for up to 180 days in the slowest tumor model ependymoma HN0579. The fastest model, high grade glioma HG0068, reached termination criteria within 24 days. Histopathological examination was strictly correlated with the tumor load determined by optical imaging in situ and ex vivo (organ imaging). The histopathological investigation of the mouse brains displayed no differences in tumor localization, size, and invasiveness between the transduced and the un-transduced lines. The proliferation rate determined by Ki-67 staining was not influenced by the modification of the cells. Further molecular and phenotypic characterization of the transduced vs the un-transduced PDX will increase the utility of this platform for the development of new drugs and the identification of innovative drug targets.

Citation Format: Eva Oswald, Kanstantsin Lashuk, Johannes Gojo, Dorothee Lenhard, Norman Mack, Sonja Krausert, Daniela Lötsch, David Jones, Marcel Kool, Till Milde, Walter Berger, Stefan M. Pfister, Julia Schüler. Establishment and characterization of pediatric brain tumor models in an orthotopic mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1673.
Abstract 1694: Cytotoxic conditions alter cell free DNA concentration and fragment size in cancer cells
Authors: Bustamante Prisca , Tsering Thupten , Burnier Julia Valdemarin
Published: June 2022

Introduction: Detection of circulating free DNA (cfDNA) is a promising tool to monitor and predict tumor progression and treatment efficacy. Our group has shown the clinical relevance of cfDNA as a biomarker of tumor development and progression in several cancer models, including colorectal carcinoma and melanoma. cfDNA may stem from a combination of apoptosis, necrosis, and cellular secretions. However, the source and mechanism of cfDNA release in cancer cells are still unclear. Indeed, while some data state that cfDNA is derived from apoptosis, other studies report it is mainly a product of cellular secretions. To better understand cfDNA release and its correlation with tumor biology, this study aimed to assess cfDNA kinetics under cell death, cell cycle arrest, and senescence by using an in vitro cancer model.

Methods: Colorectal (HTC116 and HT29), lung (A549), and melanoma (MP41 and OMM2.5) cancer cells underwent cytotoxic conditions by drugs: TRIAL/APO2L, Roscovitine, and valproic acid (VAP), as well as environmental stress by heat and irradiation. Cell death and cell cycle were evaluated by FACS. β-galactosidase staining assessed senescence. cfDNA was extracted from 3 mL supernatant using QIAamp nucleic acid kit and evaluated by digital droplet PCR through hot spot mutations (Mut), and a mitochondrial gene (mt-cfDNA). Isolated cfDNA was visualized by fragment size using Bioanalyzer 2100.

Results: Mut and mt-cfDNA increased during cytotoxic conditions in a dose-dependent manner. Cells under apoptosis released greater mut and mt-cfDNA compared to necrotic and untreated cells. Similarly, cell cycle disturbances by VAP were associated with an increase in Mut and mt-cfDNA. Moreover, β-galactosidase positive cells (5D post-irradiation) showed lower Mut and mt-cfDNA levels than control conditions. Electropherogram images showed that cytotoxic conditions alter fragment size distribution. While fragments about 100-200 bp were observed in apoptosis, necrosis showed more abundant fragments about >1000 bp.

Conclusions: The release of cfDNA is influenced by cytotoxic and stress conditions. Knowing the biology of cfDNA can help understand its role in cancer development and how best to utilize this marker clinically, especially post cytotoxic agents that induce different cell death mechanisms. These findings have major implications in the interpretation of ctDNA in liquid biopsy as a biomarker of treatment response and tumor progression.

Citation Format: Prisca Bustamante, Thupten Tsering, Julia Valdemarin Burnier. Cytotoxic conditions alter cell free DNA concentration and fragment size in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1694.
Abstract 1698: Copy number diversity within and across tumor types
Authors: Baker Toby , Dentro Stefan , Spellman Paul , Tarabichi Maxime , Van Loo Peter
Published: June 2022

Introduction Cancers commonly accrue copy number gains and losses during their development. An improved understanding of their contribution to tumorigenesis can be gained by studying their distribution and evolution within and across tumor types.

Materials and Methods We analyzed copy number profiles of 16,765 tumours from the Hartwig Medical Foundation, Pan-Cancer Analysis of Whole Genomes (PCAWG) and The Cancer Genome Atlas (TCGA) cohorts. We used a novel timing method, ComplexTiming, to time the occurrence of copy number gains, and a permutation-based approach to assess the distribution of copy number events relative to a null distribution.

Results Complex copy number states in cancers that have undergone a whole-genome duplication (WGD) are commonly assumed to develop through a most parsimonious route, where the series of events leading to the complex copy number state requiring the least number of steps, is proposed to be the most likely one. Our newly developed ComplexTiming approach allows unique insights into the actual routes that led to these complex copy number states. In our large pan-cancer cohort, we found that the most parsimonious set of events does not occur in approximately 40% of cases. Additional gains more frequently occurred after WGD than before, reflecting a previously observed increase in instability. In squamous cell lung cancer, gains of 3q were frequently found before WGD. Additionally, 5q and 1q were significantly more likely to be gained before WGD in colorectal and melanoma metastases, respectively.

Evaluating the distribution of copy number events, we found that the majority of copy number events are distributed across tumors consistent with a permutation model that assigns copy number events randomly. However, interestingly, in the majority of cancer types, we found copy number events that occurred more often than expected in individual tumors with low numbers of events, such as loss of chromosome 9 in bladder cancer, loss of 20p in colorectal adenocarcinomas, and gain of 1q in breast adenocarcinomas. These results suggest that the high-frequency of these specific copy number events is due to selection, while other copy number changes may occur neutrally due to innate chromosome instability.

Conclusion The landscape of copy number gains in tumors is diverse. By analyzing the timing of these events as well as their distribution, we identify a subset of frequent copy number changes in different cancer types that are likely under selection.

Citation Format: Toby Baker, Stefan Dentro, Paul Spellman, Maxime Tarabichi, Peter Van Loo. Copy number diversity within and across tumor types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1698.