bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2618: Tumor multicomponent targeting nanoparticle for synergistic killing of drug resistant renal cell carcinoma
Authors: Vanamala Kushal , Sau Samaresh , Naik Shivani , Rishi Arun K. , Iyer Arun
Published: September 2020

Targeted therapy in renal cell carcinoma (RCC) has limited clinical response due to the development of drug resistance. Drug resistance can be acquired through multiple pathways including, impairment of apoptosis, overexpression of hypoxia promoting carbonic anhydrase 9 (CA9), and infiltration of folate receptor (FR) positive tumorigenic macrophages. Our hypothesis was to overcome the drug resistance by developing a tumor stimuli-responsive nanoparticle (NP) that can carry combination drugs, such as Cabozantinib (CB) and CFM 4.16 in RCC. The rationale of choosing the CB in combination with CFM4.16 was to induce synergistic anti-RCC effect by modulating the multiple tumor survival pathways, such as caspase 3, Akt/p-Akt, MET. One of the major challenges of the CB and CFM4.16 combination therapy is their poor water solubility and limited targetability to cancer. Thus, we came up with a smart strategy of developing dual folate receptor (FR) and carbonic anhydrase-9 (CA9) biomarker directed nanoparticles that can target the tumor stroma and hypoxia of RCC, respectively. In this proof of concept study, we synthesized SMA-TPGS and SMA polymer (PL) comprising of FR and CA9 dual targeting ligand, namely, FR-CA9-OL using reagent free ‘click' chemistry. FR-CA9-PL was encapsulated either with CFM4.16 or with CB to obtain nanoparticles, called FR-CA9-C4.16 and FR-CA9-CB respectively. The nanoparticles were prepared by using a co-solvent dialysis method that resulted in small-particle size with a range of 80-100 nm diameter, narrow polydispersity index of ∼ 0.15 and high drug loading of 30% (wt./wt.). The homogenous and high drug loading characteristics of nanoparticles are optimal for penetrating hypoxia of RCC. The IC50 of FR-CA9-C4.16 was improved by 60-fold as compared to CFM4.16 treatment and IC50 of FR-CA9-CB is improved by 16-fold compared to CB treatment in aggressive Everolimus-resistant (Evr-res) A498 RCC cells. The combination of FR-CA9-C4.16 with FR-CA9-CB demonstrated synergistic killing of Evr-res-A498 with CI value 0.1-0.5. The signature characteristic and superior anticancer effect of nanoparticles in Evr-resistant RCC provide a worthwhile strategy for overcoming drug resistance in RCC.

Citation Format: Kushal Vanamala, Samaresh Sau, Shivani Naik, Arun K. Rishi, Arun Iyer. Tumor multicomponent targeting nanoparticle for synergistic killing of drug resistant renal cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2618.
Abstract 2646: CD39+PD-1+CD8+ T cells mediate metastatic dormancy in breast cancer
Authors: de Lara Paulino Tallón , Cuadrado Héctor Castañón , Vermeer Marijne , Núñez Nicolás , Urdínez Joaquín , Cecconi Virginia , Attar Farkhondeh Movahedian , Glarner Isabelle , Hiltbrunner Stefanie , Tugues Sonia , Becher Burkhard , van den Broek Maries
Published: September 2020

Although metastatic disease can be present already at diagnosis, in some patients it appears months or years after resection of the primary tumor. This is thought to be due to the ability of some tumor cells to remain dormant after metastatic seeding. It is not known how dormancy is controlled, but there is evidence for tumor cell-intrinsic and -extrinsic mechanisms including immune defense.

Here we found in preclinical models of breast cancer that metastatic dormancy was fully controlled by T cells. Through high-parametric single-cell mapping, we identified a discrete population of CD39+PD-1+CD8+ T cells present both in primary tumors and in spontaneous dormant metastasis, whereas this population is hardly found in aggressively metastasizing tumors. Surprisingly, despite expressing many exhaustion markers, this population was rich in effector molecules and cytokines that induced dormancy in tumor cells. Of note, the adoptive transfer of purified CD39+PD-1+CD8+ T cells prevented metastatic outgrowth.

Thus, we discovered that primary breast tumors prime CD39+PD-1+CD8+ T cells that induce metastatic dormancy in distal disseminated tumor cells in the lungs.

Citation Format: Paulino Tallón de Lara, Héctor Castañón Cuadrado, Marijne Vermeer, Nicolás Núñez, Joaquín Urdínez, Virginia Cecconi, Farkhondeh Movahedian Attar, Isabelle Glarner, Stefanie Hiltbrunner, Sonia Tugues, Burkhard Becher, Maries van den Broek. CD39+PD-1+CD8+ T cells mediate metastatic dormancy in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2646.
Abstract 2656: Myeloid derived Wnt5a influences the immune-dynamics of melanoma tumor microenvironment
Authors: Douglass Stephen , Fane Mitchell , Sanseviero Emilio , Gabrilovich Dmitry , Weeraratna Ashani
Published: September 2020

Despite breakthroughs in targeted therapies and immunotherapies, over 75% of patients either do not respond to therapy, or relapse. This may be due in part to changes in their tumor microenvironment (TME). Wnt5A is a glycoprotein that has been shown to influence melanoma phenotype, promoting a slow-cycling, but more invasive cancer. Wnt5A was thought to be produced from tumor cells and fibroblasts in the TME. However, our data now suggest that unique pathologically induced myeloid derived suppressor cells (MDSCs) produce a major proportion of Wnt5A in the TME which is taken up by melanoma cells to increase invasiveness. This finding highlights the diverse roles that MDSCs play in melanoma, going beyond immunosuppression. We created novel transgenic animals with MDSC specific knockdown of Wnt5A and demonstrate a clear decrease in the Wnt5A expression within the TME. Wnt5A deficient MDSCs caused an increase in CD8+ T-cells in peripheral blood and decreased tumoral MDSCs and Tregs. Tumor infiltrating lymphocytes also had significantly decreased PD-1 expression suggesting a less exhausted phenotype, and increased ratios of effector T-cells to immunosuppressive cells resulting in a more immunogenic TME. Wnt5A promotes a switch from proliferative to an invasive melanoma cell state. Myeloid specific Wnt5A knockdown caused decreased lung metastasis and significantly increased tumor growth in vivo, with the lack of MDSC infiltration negatively correlating to tumor volume. Tumor infiltrating MDSCs from control animals had a strong positive correlation with Tregs, whereas, this was completely ablated in animals with Wnt5A-negative MDSCs. Overall, our data suggests that while MDSCs contribute to an immunosuppressive and less immunogenic environment, they may have an additional function as the major source of Wnt5A in the tumor microenvironment. These two functions likely synergize to drive a highly metastatic and therapy-resistant phenotype altering immunogenicity.

Citation Format: Stephen Douglass, Mitchell Fane, Emilio Sanseviero, Dmitry Gabrilovich, Ashani Weeraratna. Myeloid derived Wnt5a influences the immune-dynamics of melanoma tumor microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2656.
Abstract 2713: Elucidation of bone metastasis using a caudal artery injection mouse model and Akaluc-AkaLumine near-infrared bioluminescence technology
Authors: Kuchimaru Takahiro , Minegishi Misa , Kizaka-Kondoh Shinae
Published: September 2020

Bone is one of the most common sites of metastasis for various primary tumors including breast, prostate and lung cancers. We have addressed molecular basis underlying bone metastasis using newly developed murine model of bone metastasis and near-infrared bioluminescence technologies. Our new murine model overcomes several limitations in the current standard model by intra-cardiac (IC) injection of cancer cells [1]. We found that the caudal artery is a new pathway to predominantly deliver cancer cells to the bone marrow of hind limbs in mice. Intra-caudal arterial (CA) injection is technically as easy as tail vein injection [2]. In addition, CA injection delivered cancer cells >10-fold more efficiently than IC injection. This accelerated the development of bone metastasis in hind limbs with wide variety of cell lines. We have combined the CA injection model with near-infrared bioluminescence imaging. Bioluminescence imaging (BLI) with firefly luciferases and its natural substrate D-luciferin has been indispensable to noninvasively monitor metastasis development in mice. To improve deep-tissue imaging, synthetic luciferin AkaLumine has successfully extended the wavelength of firefly bioluminescence to the near-infrared region where tissue is more transparent to light [3]. More recently, our collaborators created an engineered luciferase Akaluc that displays high catalytic activity in reaction with AkaLumine [4]. AkaLumine-Akaluc bioluminescence imaging (AkaBLI) is capable of noninvasive visualization of single cancer cells deep inside mice. Combining the CA injection model with AkaBLI technology is a powerful tool for elucidating the mechanism of bone metastasis in small animals. References [1] Kuchimaru et al. Nat Commun 2018, 9, 2918 [2] http://sites.google.com/view/takahirokuchimaru/protocol [3] Kuchimaru et al. Nat Commun 2016, 7, 11856 [4] Iwano et al. Science 2018, 359, 935

Citation Format: Takahiro Kuchimaru, Misa Minegishi, Shinae Kizaka-Kondoh. Elucidation of bone metastasis using a caudal artery injection mouse model and Akaluc-AkaLumine near-infrared bioluminescence technology [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2713.
Abstract 2726: Nuclear checkpoints for melanoma and breast cancer lung metastasis
Authors: Roncato Francesco , Regev Ofer , Levi Nehora , Feigelson Sara W. , Gerlitz Gabi , Alon Ronen
Published: September 2020

Introduction

Metastasis involves cancer cell intravasation and extravasation from blood vessels at target organs. The mechanisms by which the nuclei of metastatic tumor cells squeeze through different vascular and epithelial barriers are poorly understood. The nuclear lamina attaches chromatin domains to the nuclear periphery and controls the mechanical properties of the nucleus and its crosstalk with the cell cytoskeleton. A-type lamins, lamin A and its splice variant lamin C, are key nuclear lamina proteins that control nucleus stiffness and regulate chromatin conformation. Reduced lamin A was reported in some aggressive cancers.

Results and Discussion

Using a new in vitro transendothelial migration (TEM) assay we found that melanoma and breast cancer cells slowly squeeze their nuclei and complete TEM through endothelial junctions irrespective of their relative lamin A/C levels, suggesting that lamin A/C dependent nuclear stiffening is not a rate-limiting step for cancer cell squeezing through endothelial cells in vitro. In contrast, reduced lamin A/C enhanced cancer cell squeezing through rigid pores. Tumor emigration analysis across blood vessels in vivo was performed by new 3D imaging of fluorescently labeled cancer cells with high and low lamin A/C levels. This imaging was based on light sheet microscopy of lipid-cleared lungs of recipient mice in situ labeled with anti-vascular CD31 mAb. Using this technology, we found that reduced lamin A/C expression did not enhance the emigration of circulating murine melanoma and breast cancer cells across lung capillaries in vivo. Lower lamin A/C levels reduced the constitutive, transcriptionally repressed, heterochromatin in both melanoma and breast cancer cells. Decreased lamin A/C expression did not enhance however, nuclear rupture in the tumor cells that entered the lung parenchyma. The outcomes of the epigenetic and mechanical changes resulting of lamin A/C suppression on tumor growth and metastasis are under current investigation.

Conclusions

Our results suggest that subsets of tumor cells may benefit from reducing their nuclear content of lamin A/C mainly at the level of rigid barrier crossing (e.g. dense interstitial spaces), but not at the level of extravasation or post extravasation survival at target organs. How alterations in lamin A/C content contribute to tumor heterogeneity and metastatic potential of singular cancer cells remains an open question.

Citation Format: Francesco Roncato, Ofer Regev, Nehora Levi, Sara W. Feigelson, Gabi Gerlitz, Ronen Alon. Nuclear checkpoints for melanoma and breast cancer lung metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2726.
Abstract 2730: BCL6 promotes ovarian cancer metastasis
Authors: Nguyen Duc Ngoc Hong , Walker David F. , Frank David A. , Walker Sarah R.
Published: September 2020

Ovarian cancer remains a deadly disease with the five-year survival rate for ovarian cancer patients at about 50%. Ovarian spheroids, clusters of 3D growing cells, are thought to promote ovarian cancer metastasis and serve as a reserve of cancer cells that are not responsive to chemotherapy. Understanding genes associated with spheroid growth and metastasis may identify pathways to target for ovarian cancer therapy. We have found that the transcriptional modulator BCL6 is active in ovarian spheroids. BCL6 is a highly-conserved oncogene originally found to drive malignant transformation in B-cell lymphoma. Although BCL6 is thought to be a transcriptional repressor, we and others have found that it also upregulates genes. Little is known about the role of BCL6 in ovarian cancer. Using the Cancer Genome Atlas (TCGA) data and CBioPortal, we found that BCL6 is amplified in about 20% of high-grade serous ovarian cancer patients. In addition, analysis of nuclear extracts from a number of ovarian cancer cell lines demonstrated that BCL6 is overexpressed in many ovarian cancer cell lines. To assess transcriptional activity, we used a BCL6-responsive reporter and we found that BCL6 was active in most of these cell lines. Having demonstrated that BCL6 is active in ovarian cancer cells, we wanted to determine if BCL6 affected the viability of these cells. Therefore, we transfected ovarian cancer cells with siRNA to BCL6, measured their viability, and found that BCL6 is important for ovarian cancer cell survival. Recognizing that spheroids occur in advanced ovarian cancer, we analyzed the effects of BCL6 in 3D formation using micropatterned plates and discovered that BCL6 is also important for 3D spheroid formation. A major problem in advanced ovarian cancer is metastasis; therefore, we wanted to determine if BCL6 plays a role in promoting metastasis. We overexpressed BCL6 or downregulated BCL6 with siRNA and performed a mesothelial clearance assay to assess the early steps in ovarian cancer metastasis. While overexpressing BCL6 enhanced mesothelial clearance by the ovarian spheroids, reducing BCL6 expression also reduced mesothelial clearance. This suggested that BCL6 promotes ovarian cancer metastasis. We are currently in the process of identifying BCL6 target genes that are involved in metastasis. Taken together, our data suggests that BCL6 is an important transcriptional modulator in ovarian cancer and that targeting BCL6 in the future may be a useful strategy to treat metastatic ovarian cancer.

Citation Format: Duc Ngoc Hong Nguyen, David F. Walker, David A. Frank, Sarah R. Walker. BCL6 promotes ovarian cancer metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2730.
Abstract 274: Multifaceted function of microRNA-299-3p fosters an antitumor environment through modulation of androgen receptor and VEGFA signaling pathways in prostate cancer cells
Authors: Ganapathy Kavya , Staklinski Stephen , Hasan Md Faqrul , Ottman Richard , Andl Thomas , Berglund Anders , Park Jong , Chakrabarti Ratna
Published: September 2020

MicroRNAs (miRNAs) play crucial roles in the regulation of prostate cancer (PCa) through modulation of signaling pathways. Androgen receptor (AR) signaling is central to PCa and PCa therapy. The therapeutic suitability of miRNAs that target AR has not been investigated thoroughly. Here, we illustrate the clinical relevance, functional significance and therapeutic benefit of miR-299-3p, an AR targeting microRNA, in PCa progression. We noted a loss of expression of miR-299-3p in prostate tumors compared to noncancerous prostate tissue in clinical specimens. Studies on the impact of miR-299-3p restoration on AR function, phenotypic changes associated with cancer progression and involvement of the downstream components of the AR signaling pathway were conducted using in vitro and in vivo model systems. Replenishment of miR-299-3p in C4-2B, 22Rv-1, and PC-3 cells contributed to cell cycle arrest, reduced cell proliferation, migration and increased expression of apoptotic markers. Additionally, expression of miR-299-3p reduced AR, PSA and VEGFA expression. AGO-RNA pulldown experiment showed enrichment of AR, VEGFA and miR-299-3p in the RISC in the extracts of C4-2B cells overexpressing miR-299-3p. Overexpression of miR-299-3p inhibited epithelial-mesenchymal transition signaling, through inhibition of expression of Slug, TGF-Β3, phospho-AKT, and phospho-PRAS40 and up-regulation of E-cadherin. Replenishment of miR-299-3p showed reduced tumor growth in xenograft mice models. Furthermore, we also tested the benefit of miR-299-3p restoration in combination treatment with enzalutamide and docetaxel which showed a synergistic effect on drug sensitivity. In conclusion, this study has identified novel mechanisms of antitumor and antimigration functions of miR-299-3p through modulation of AR and VEGFA signaling pathways which leads to improved drug sensitivity of PCa.

Citation Format: Kavya Ganapathy, Stephen Staklinski, Md Faqrul Hasan, Richard Ottman, Thomas Andl, Anders Berglund, Jong Park, Ratna Chakrabarti. Multifaceted function of microRNA-299-3p fosters an antitumor environment through modulation of androgen receptor and VEGFA signaling pathways in prostate cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 274.
Abstract 2750: An efficient multiplexing approach to avoid batch effects in single cell RNA- and Ab-seq studies demonstrated in a mouse model of chronic inflammation and obesity
Authors: Song Hye-Won , Baracho Gisele , Taylor Ian , Widmann Stephanie , Mortimer Stefanie
Published: September 2020

Obesity can weaken the body's immune system and reduce its ability to fight off infections. Current analysis on immune cells from obese mouse models suggests that the immune cells suffer from chronic inflammation. To understand the root cause of this problem, we used single cell sequencing to examine thousands of immune cells isolated from primary and secondary lymphoid organs as well as adipose tissue and compared a diet-induced obesity mouse model with control mice. Cells were stained with 30 BD® AbSeq DNA-barcoded antibodies to enable multiomic analysis, i.e. examining protein alongside mRNA expression in tandem. We also utilized DNA-barcoded universal antibodies from the BD® Single-Cell Multiplexing Kit, which allowed us to combine 8 samples from different mice and tissue types into a single pooled sample, significantly reducing experimental scale and cost while eliminating potential batch effects. The pooled samples were loaded on the BD Rhapsody™ system to perform cell lysis and individual mRNA and cell barcoding, allowing measurement of ~400 immune-related mRNAs and 30 proteins at the single cell level. We were able to efficiently de-multiplex the pooled samples after sequencing. The targeted mRNA and AbSeq panel provided robust clustering of immune cell types and showed that genes related to critical immune responses, including inflammation and lymphocyte activation, are differentially regulated in specific immune-cell subsets in the obese mouse model. Using this multiomic analysis of genes differentially regulated in immune cells from different tissues, we propose a model to explain the immuno-phenotype we observed in obese mouse.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.

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Citation Format: Hye-Won Song, Gisele Baracho, Ian Taylor, Stephanie Widmann, Stefanie Mortimer. An efficient multiplexing approach to avoid batch effects in single cell RNA- and Ab-seq studies demonstrated in a mouse model of chronic inflammation and obesity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2750.
Abstract 2759: Immune cell killing assay for analyzing the effects of immunomodulating agents in vitro
Authors: Mäki-Jouppila Jenni H. , Halleen Jussi M. , Fagerlund Katja M.
Published: September 2020

Immuno-oncology has emerged as one of the most promising new approaches in cancer drug development. Greater cancer cell specificity of treatments can be achieved when the immune cells of a patient are used against tumors. Therefore, it is important to study the effects of immune cells on cancer cells using different models in vitro and in vivo. Our aim was to validate an in vitro T cell killing assay where activated peripheral blood mononuclear cells (PBMCs) target different cancer cells and increase their apoptosis.

MCF-7 human breast carcinoma cells (ATCC) were used as target cells and were seeded on 96-well plates. 24 hours later, human PBMCs (Lonza) were added to the culture as effector cells. Simultaneously, the PBMCs were activated with 100 ng/ml anti-CD3 and 10 ng/ml IL2. Apoptosis was detected using IncuCyte Annexin V reagent, which was introduced to the cells at the same time with activators. Cell growth, proliferation and apoptosis were monitored with a live cell imager IncuCyte S3 (Sartorius) for three days. Cell viability was determined by CellTiter Glo (Promega) at the end of the study.

The target-to-effector ratio of 1:5 and 1:10 both showed an increase in apoptosis of MCF-7 cells in the wells with activated PBMCs. This was detected with a confluence parameter (percentage) of IncuCyte S3.

The T cell killing assay can be utilized for studying cancer therapeutics based on immune-oncology (e.g. pembrolizumab and atezolizumab) and to evaluate the potential of immune cells in killing cancer cells in the presence of drugs affecting the target-effector interaction.

Citation Format: Jenni H. Mäki-Jouppila, Jussi M. Halleen, Katja M. Fagerlund. Immune cell killing assay for analyzing the effects of immunomodulating agents in vitro [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2759.
Abstract 2760: A mouse orthotopic model of pancreatic cancer and response to treatment
Authors: Rainbolt Elizabeth , Cecil-Taylor Madelyn , Wong Andrew , Ball Robin , Wood Patrick , Wood Edgar R. , de Marval Paula Miliani , Yeyeodu Susan
Published: September 2020

The mouse pancreatic ductal adenocarcinoma PAN02 was produced in C57BL/6 mice exposed to 3-methyl-cholanthrene. PAN02 is generally resistant to standard cancer therapeutics, thus providing an appropriately rigorous model for the development of clinically effective agents to treat aggressive and intractable pancreatic ductal adenocarcinoma. Subcutaneous implants of syngeneic PAN02 provide the dual advantages of being able to closely monitor changes in tumor growth without compromising the complex pro- and anti-tumor immune responses of the host that collectively determine the outcome of tumor progression. Unfortunately, subcutaneous PAN02 tends to grow slowly and tumor ulcerations are common which may result in tumor regression or loss of animals in a study. Here we report the growth properties and response to therapy of orthotopically implanted PAN02. To establish the orthotopic model, we implanted cells by direct injection into the pancreas through an incision in the abdominal wall at two implant densities, 3 × 105 and 3 × 106 PAN02 cells in 30 μl. Both cell inocula demonstrated progressive growth with median survival of thirty-seven and thirty days post implant respectively. We chose 3 × 106 PAN02 cells to evaluate efficacy of cytotoxic agents gemcitabine and oxaliplatin. Dosing was initiated 7 days after implant; gemcitabine was dosed at 120 mg/kg, by intraperitoneal injection (IP) every third day for four doses and oxaliplatin was dosed at 10 mg/kg IP once a week for 3 weeks. Gemcitabine and oxaliplatin increased life span by 9.0 days and 20.5 days respectively over vehicle control. The morphology of the tumors revealed by histological staining is also presented. The development of this orthotopic pancreatic model provides a more clinically relevant model for evaluation of novel therapies compared to subcutaneous implantation.

Citation Format: Elizabeth Rainbolt, Madelyn Cecil-Taylor, Andrew Wong, Robin Ball, Patrick Wood, Edgar R. Wood, Paula Miliani de Marval, Susan Yeyeodu. A mouse orthotopic model of pancreatic cancer and response to treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2760.
Abstract 2763: Identification of mouse embryo genes in human cancer using cBioportal
Authors: Xiong Miranda , Wang Christina , Chen Eric , Hu Arthur , Luo Raymond
Published: September 2020

Cancer is the second leading cause of death, often without a cure. There are many genetic factors that can either cause cancer, worsen existing conditions, or shorten a person's life span; however, it is not fully understood. During embryogenesis, many genes are expressed during a short window of the developmental stages and then switched off in adults. It is known that some of the embryogenesis specific genes are switched on in tumors. The purpose of this study was to identify genes that are exclusively expressed in the central nervous system (CNS) of the E14.5 mouse embryo (Dlez-Roux, G. et al) associated with cancer to provide a foundation for future research.Using cBioPortal, we queried the TCGA PanCancer Atlas Studies (32 studies and 10,967 samples) by each of the 80 genes expressed in the CNS of E14.5 mouse embryo. The data types analyzed included cancer type percentages and survival estimate (overall and disease/progression-free Kaplan-Meier estimate). To identify genes that are relevant in human cancer, the 80 genes were filtered based on three criteria: whether the gene mutations were found in at least 10% of various cancer types, whether the p-value for either the overall survival Kaplan-Meier Estimate, and of the disease/progression-free Kaplan-Meier Estimate was equal or less than 0.05. Fourteen genes carried mutations in more than 10% of various cancer types. Mutations in nine genes significantly decreased cancer patients' overall survival, but mutations in one gene significantly improved overall survival. Mutations in ten genes also significantly decreased the patients' disease/progression-free survival. Three genes demonstrated a high mutation rate (>15%) and significantly reduced patients' survival in both overall survival and disease/progression-free estimates. None of these genes have been identified as cancer driver genes. We report here that many of the CNS E14.5 mouse embryonic genes are mutated in human cancers and some of the mutations may predict cancer patient outcome. Using the same approach, we will evaluate whether these genes are correlated with the manifestation of all cancers or a subset of cancer types. In the future, we would like to obtain the opportunity to study the role of these genes in cancer development or cancer treatment in a laboratory. Dlez-Roux, G. et al: A high-resolution anatomical atlas of the transcriptome in the mouse embryo. PLoS Biol. 2011This study was supervised by Dr. Yangjun Qin, and all authors contributed equally.

Citation Format: Miranda Xiong, Christina Wang, Eric Chen, Arthur Hu, Raymond Luo. Identification of mouse embryo genes in human cancer using cBioportal [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2763.
Abstract 2776: Real-time measurement of temperature and tissue damage during laser photothermal ablation
Authors: Fathi Faraneh , Maddirela Dilip R. , Gogineni Venkateswara Rao , White Sarah B. , Yu Bing
Published: September 2020

Hepatocellular carcinoma (HCC) is the sixth most common cancer and tumor resection is feasible in only 25±30% of these patients. Thermal ablation therapies using lasers, radiofrequency waves and microwaves are considered as an alternative treatment for unresectable tumors. Our goal was to develop optical fiber-based laser ablation while monitoring the changes in temperature and tissue properties in real-time. A portable diffuse reflectance spectroscopy (DRS) system consisting of a DRS probe with self-calibration and fiber optic temperature sensor was made. Tissue mimicking phantoms using human hemoglobin powders and polystyrene particles with fixed total number of scatterers were used to calibrate the system. In vivo studies were performed in 8 rats orthotopically implanted with HCC. Temperature sensor was inserted in the center of the tumor via hypodermic needle and DRS fiber was overlaid on the temperature sensor. Ablation was performed by a fiber optic diffuser connected to an 808nm laser using different power settings in pulse mode. The tissue temperature and optical properties were continuously monitored until both plateaued using a custom LabVIEW program. Rats were immediately euthanized, and the ablated liver tissues were collected for histological analysis. The DRS showed average errors of 6.31% and 4.74% in measuring the absorption (μa) and reduced scattering coefficients (μs) of the phantoms respectively. Tumor bearing liver tissues showed a decreasing trend in both μa and μs irrespective of the laser power (1W-2.5W) and the temperatures recorded (50°C to 88°C) in different animals. Histological analysis showed discrete zones of tissue damage from treated regions with hemorrhage in the outer most zone. Changes in optical properties of the ablated tumors with temperatures and histological changes needs further correlation by performing more in vivo studies.

Citation Format: Faraneh Fathi, Dilip R. Maddirela, Venkateswara Rao Gogineni, Sarah B. White, Bing Yu. Real-time measurement of temperature and tissue damage during laser photothermal ablation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2776.
Abstract 2790: Evaluation of S100A9 targeted nanoparticles containing rapamycin to treat pancreatic adenocarcinoma
Authors: Thomas Karl , McNally Molly , Samykutty Abhilash , McNally Lacey R.
Published: September 2020

Despite Pancreatic cancer being expected to contribute to only 3% of new cancer cases in both males and females in 2019, it is expected to cause an estimated 45,750 deaths making it the third highest contributor to cancer deaths. This is due to the low survivability associated with pancreatic cancer which has a 5-year survival rate of only 9%. In order to address the poor prognosis common with pancreatic cancer, biomarkers are being used to create therapies targeted to specific cancer cells. The S100A8/A9 complex is a heterodimer, known for modulating the inflammatory response, and it is upregulated in a variety of cancers including pancreatic cancers. For this reason, we selected it as a targeting agent to deliver Rapamycin to pancreatic cancer cells via a mesoporous silica-encased gold nanorod (MS-GNR). The use of this nanoparticle allows an anticancer molecule to be encapsulated within the mesoporous silica matrix by further encapsulation of the nanoparticle with the pH sensitive polymer chitosan. A low pH tumor microenvironment induces relaxation of the chitosan surrounding this chitosan-capped mesoporous silica gold nanorod (CMG) allowing for the anticancer molecule to be released only in cancer cells. The gold nanorod at the core of this enables these nanoparticles to be imaged in real time via Multispectral Optoacoustic Tomography (MSOT). Using these tools, we linked S100A9 to a mesoporous silica-encased gold nanorod which contained Rapamycin held in via the chitosan polymer (S100A9-Rap-CMG). MSOT imaging revealed a strong uptake in S2013Q and MiaPaca pancreatic cancer cells. Further tests showed that at a pH 6.8 S100A9-Rap-CMG treatment resulted in an increase in cell death by 69% compared to treatment at physiologic pH of 7.4 by the S100A9-Rap-CMG's which did not induce cell death. This data indicates the feasibility of S100A9 as a targeting agent for pancreatic cancer cells and the potential benefits of nano-drug delivery of Rapamycin to treat pancreatic adenocarcinoma. The ultimate benefit of the nano-drug delivery includes both increased tumor targeting and mitigating premature drug release and offsite delivery.

Citation Format: Karl Thomas, Molly McNally, Abhilash Samykutty, Lacey R. McNally. Evaluation of S100A9 targeted nanoparticles containing rapamycin to treat pancreatic adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2790.
Abstract 2824: In vitro tumor killing assays for predicting cellular therapeutic efficacy
Authors: Nunan Robert , Yap Michelle , Garcia-Alvarez Enrique , Carkill Marie , Pascoe Anna , Brackenbury Louise , Self-Fordham Jezrom , Jenkinson S. Rhiannon
Published: September 2020

Cellular immunotherapy is emerging as a fifth pillar of cancer treatment. Clinical efficacy has been proven for a variety of cancers; however, its true potential has yet to be unlocked. Development of reliable and mechanistically appropriate in vitro screening assays that recapitulate in vivo immune-tumor interactions have been outpaced by in vivo living cell therapy efficacy models. Here we describe a range of in vitro primary human assays to model tumor killing by immune cells. This platform addresses multiple anti-tumor immune effector pathways and has been validated with standard of care therapeutics. Fluorescently labelled target cells (PDX-cell lines, tumor cell lines or human primary/iPSC derived cells) screened for relevant antigen expression were cultured in monolayer or spheroid forms and co-cultured with the cellular therapeutic. On target killing by effectors is driven by binding of the TCR or CAR to its cognate antigen (MHC/peptide or protein, respectively) with readouts of cell killing utilising live cell imaging (IncuCyte) and flow cytometry based approaches to measure target cell growth and apoptosis. Additional analysis of soluble mediators can determine the extent of effector cell activation. To discriminate between ‘on' and ‘off' target killing by cellular therapeutics, primary/iPSC-derived normal cell types representative of different tissue types were screened for expression of an HLA subtype capable of binding exogenously applied tumor-associated antigen (TAA) prior to co-culture. Under these conditions in the case of a TCR based cellular therapeutic, efficacy can be screened for, or in the absence of the putative TAA expression, the extent of off-target killing determined. Enhancement of tumor targeting and killing by immune cells is an attractive therapeutic modality partly due to the clinical success of checkpoint inhibitors which can enhance the activation and effector function of endogenous T/NK cells or cellular therapeutics. The presented data highlight the importance of assays which can recapitulate tumor-immune interactions and include immune effector pathways known to potentiate anti-tumor immune responses. These assays represent valuable screening tools capable of informing cellular therapeutic target selection and refinement before moving to more complex in vivo models.

Citation Format: Robert Nunan, Michelle Yap, Enrique Garcia-Alvarez, Marie Carkill, Anna Pascoe, Louise Brackenbury, Jezrom Self-Fordham, S. Rhiannon Jenkinson. In vitro tumor killing assays for predicting cellular therapeutic efficacy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2824.
Abstract 2898: A biparatopic HER2xHER2 antibody drug conjugate with potent anti-tumor activity
Authors: Bay Andres Perez , Faulkner Devon , Tiwari Shiwani , Castanaro Carla , Zheng Xiang , Kunz Arthur , Nittoli Thomas , Han Amy , Olson William , Thurston Gavin , Daly Christopher , Andreev Julian
Published: September 2020

A biparatopic HER2xHER2 antibody drug conjugate with potent anti-tumor activity.

The antibody drug conjugate (ADC) Trastuzumab-emtansine (T-DM1) targets the well-characterized breast cancer oncogene HER2 and is active in tumors that express very high levels of HER2 protein. However, tumors expressing intermediate levels of HER2 remain resistant to T-DM1 therapy, apparently due to insufficient lysosomal trafficking of T-DM1. We generated a series of biparatopic HER2xHER2 antibodies in which each arm recognizes a distinct epitope of HER2. One of our HER2xHER2 antibodies bound to cells with greater avidity than Trastuzumab and formed visible cell surface clusters that were efficiently internalized. While some of the internalized HER2xHER2 antibody trafficked to lysosomes and promoted HER2 degradation, a significant fraction recycled back to the plasma membrane through recycling endosomes. To exploit the increased internalization and recycling of the HER2xHER2 antibody, ADCs were generated by site-specific conjugation of Tubulysin A payload through a protease-cleavable linker. Recent data suggest that cleavage of this linker does not require lysosomal trafficking, but can occur in recycling endosomes as well. HER2xHER2-Tubulysin killed cell lines expressing high (IHC3+) and intermediate (IHC2+) HER2 levels with subnanomolar IC50, while exhibiting very weak killing of cells expressing low (IHC1+) HER2 levels (IC50 greater than 10nM). HER2xHER2-Tubulysin induced complete and durable tumor regression and outperformed T-DM1 in a collection of IHC3+ and 2+ tumor xenograft models (including PDX). Our work shows that a detailed understanding of antibody trafficking can enable the design of more effective ADCs and suggests that HER2xHER2-Tubulysin is a potential clinical candidate for HER2 IHC2+ breast cancer.

Citation Format: Andres Perez Bay, Devon Faulkner, Shiwani Tiwari, Carla Castanaro, Xiang Zheng, Arthur Kunz, Thomas Nittoli, Amy Han, William Olson, Gavin Thurston, Christopher Daly, Julian Andreev. A biparatopic HER2xHER2 antibody drug conjugate with potent anti-tumor activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2898.
Abstract 291: Functionalizing novel modulators in lung cancer
Authors: Lee Yi Fei , Yuan Ju , Lim Edwin , Chen Jianbin , Zhai Weiwei , Tan Nguan Soon , Tam Wai Leong
Published: September 2020

Lung cancer is the leading cause of cancer mortality globally, with lung adenocarcinoma being the most prevalent subtype. The discovery of driver mutations has paved the way for the use of targeted therapy, notably EGFR inhibitors, in the management of advanced disease. However, the effectiveness of targeted therapy is limited firstly by the eventual development of treatment resistance, and secondly by the sizeable percentage of cases in which the driver mutations remain unknown. Furthermore, lung adenocarcinoma (LUAD) in Asians is known to exhibit a molecular profile distinct from that in Caucasians, yet the majority of existing cohort studies have been focused on the latter. To address this gap, we and our collaborators have spearheaded the largest Asian LUAD cohort study, comprising genomic and transcriptomic data from 305 patients. The MutSigCV and 20/20+ driver prediction algorithms were applied to whole exome sequencing data from our cohort and yielded seven novel candidate drivers that had not been previously implicated in LUAD. We proceeded to functionally validate these drivers by overexpressing or knocking them down in patient-derived transformed lung cell lines and observing their effects on tumorigenicity via a number of in vitro and in vivo tumorigenicity assays. We have identified a poly (ADP-ribose) polymerase (PARP) gene, which was mutated at a relatively high frequency within our cohort, and show that PARP is crucial for modulating the tumorigenicity of lung cancer cells. In LUAD patients, reduced PARP expression levels were correlated with poorer prognosis, suggesting a putative tumor suppressor role. We further observed a loss of PARP copy number both within our cohort as well as the Caucasian The Cancer Genome Atlas (TCGA) LUAD cohort. We are now working to dissect the underlying mechanisms and explore PARP's clinical utility. This work illustrates the use of a platform to systematically model genetics in patient-derived models to study novel driver functions.

Citation Format: Yi Fei Lee, Ju Yuan, Edwin Lim, Jianbin Chen, Weiwei Zhai, Nguan Soon Tan, Wai Leong Tam. Functionalizing novel modulators in lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 291.
Abstract 293: Heat shock factor 1 directly suppresses AMP-activated protein kinase to promote tumorigenesis
Authors: Su Kuo-Hui , Dai Siyuan , Tang Zijian , Xu Meng , Dai Chengkai
Published: September 2020

Heat shock factor 1 (HSF1), a master transcriptional regulator of the evolutionarily conserved proteotoxic stress response, preserves proteomic stability upon environmental insults. HSF1 is also a potent pro-oncogenic factor, suggesting that proteomic stability enables oncogenesis. AMP-activated protein kinase (AMPK), a key cellular metabolic sensor and downstream effector of the tumor suppressor LKB1, activates catabolic pathways but suppresses anabolic pathways, including fatty acid and cholesterol biosynthesis, thereby maintaining energy homeostasis. We previously showed that upon activation by metabolic stressors, AMPK interacts with and phosphorylates HSF1 to inactivate it and cause proteomic instability, leading to tumor suppression. Intriguingly, we also discovered that HSF1 suppresses AMPK and controls body fat mass. This study is to investigate the mechanisms by which HSF1 reciprocally suppresses AMPK and the roles of this suppression in lipid metabolism and tumorigenesis. By utilizing recombinant proteins, HSF1 peptide libraries and transcription-deficient HSF1 mutants, our in vitro studies reveal that through physical interactions HSF1 imposes multilayer regulations on AMPK, including blocking AMP binding to γ subunits, impairing LKB1-mediated Thr172 phosphorylation, promoting Thr172 de-phosphorylation by PP2A, and impeding ATP binding to catalytic α subunits. Results from circular dichroism spectroscopy reveal that HSF1 induces global AMPK conformational changes. Biologically, Hsf1 deficiency suppresses lipogenesis and decreases lipid content via AMPK activation. Moreover, Hsf1-deficient cells and mice display reduced cholesterol levels. Interestingly, this defect leads to impaired cholesteroylation of sonic hedgehog (SHH). Consequently, the oncogenic SHH signaling is impaired in HSF1-deficient cells. In vivo, Hsf1 deficiency reduces body fat mass in mice, which can be markedly rescued by either pharmacological or genetic inhibition of AMPK. Importantly, the transcription-deficient HSF1 mutant, through AMPK suppression, enhances the lipid content and SHH cholesteroylation, and promotes the in vivo growth of xenografted human melanomas. In conclusion, HSF1 is a direct AMPK antagonist. This transcription-independent interaction of HSF1 with AMPK epitomizes a reciprocal kinase-substrate regulation whereby lipid metabolism and proteomic stability intertwine and promotes cancer anabolism and oncogenesis.

Citation Format: Kuo-Hui Su, Siyuan Dai, Zijian Tang, Meng Xu, Chengkai Dai. Heat shock factor 1 directly suppresses AMP-activated protein kinase to promote tumorigenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 293.