bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1560: Elucidating the differential regulation of novel long non coding RNAs and their mechanism of action in p73 dependent manner
Authors: Bareja Chanchal , Uboveja Apoorva , Saluja Daman
Published: June 2022

INTRODUCTION: The p53 tumor suppressor family is classically activated after DNA damage and plays a central role in cell fate decisions. Although, the p53 family activates many of the same genes in response to DNA damage, p73 plays distinct biological functions in development and metastasis. It is likely that p73 activates a unique transcriptional network which is critical for its anti-metastatic and anti-invasive action. Long non-coding RNAs (lncRNAs) are a class of mRNA-like transcripts longer than 200 nucleotides. They lack protein-coding ability and are believed to be involved in various kinds of biological processes. Increasing evidence suggests that lncRNA are frequently aberrantly expressed in cancers. Therefore, the roles of dysregulated functional lncRNA in human malignant tumors have attracted considerable scientific interest. The objective of our study is to find out novel long non-coding RNAs that can act as transcriptional targets of p73 and to delineate their role in p73-mediated anti-metastatic response.

METHODS: For this purpose, we performed transcriptome sequencing in HCT116p73wt and HCT116p73KD cells and screened the data for modulation of expression of lncRNAs in differential manner. Quantitative Real Time PCR was further carried out to validate the data obtained after screening RNA seq Data. Promoter analysis was carried out for the identification of p73 binding sites in the selected upregulated or downregulated lncRNAs which was further confirmed by Luciferase reporter, ChIP and site directed mutagenesis assays.

RESULTS: About six lncRNAs were observed to be significantly upregulated while four were down-regulated upon knockdown of p73. The promoters of selected lncRNAs were analysed in silico using TF Bind and JASPAR software for p73 binding sites and luciferase reporter assays suggested regulation of lncRNAs by p73. Chromatin immunoprecipitation showed promoter enrichment of the selected lncRNAs. Site directed mutagenesis further confirmed the exact binding sites of p73 onto the promoters of these novel long non coding RNAs.

CONCLUSION: Together, our study provides insights into the differential regulation of long non-coding RNAs in p73 dependent manner which further will provide the mechanism of their action at the genome level.

Citation Format: Chanchal Bareja, Apoorva Uboveja, Daman Saluja. Elucidating the differential regulation of novel long non coding RNAs and their mechanism of action in p73 dependent manner [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 1560.
Abstract 1561: Lnc-NR2F1 upregulated by a ZEB1/NR2F1 axis promotes the migration and invasion of lung cancer cells via TWIST2 regulation
Authors: Kim Eun Ju , Ahn Young-Ho
Published: June 2022

Long non-coding RNAs (lncRNAs) are RNA transcripts longer than 200 nucleotides lacking protein-coding potential, which are involved in diverse pathophysiologic conditions including cancer progression and metastasis. Metastasis is initiated by a cellular process, epithelial-to-mesenchymal transition (EMT), and many lncRNAs can be affected by and participate in the EMT process. To find out lncRNAs regulated by the EMT signals, we performed an NGS-based RNA sequencing in murine lung cancer cells, and finally selected lnc-NR2F1 that is upregulated by ZEB1, an EMT-inducing transcription factor. lnc-NR2F1 expression levels showed a positive correlation with ZEB1 mRNA levels and EMT scores in 13 mouse lung cancer cells or in lung adenocarcinoma data from TCGA. The migration and invasion of lung cancer cells were decreased after lnc-NR2F1 knockdown and increased after lnc-NR2F1 overexpression. lnc-NR2F1 was transcriptionally up-regulated by NR2F1, a transcription factor transcribed from the antisense strand. Interestingly, NR2F1 was transcriptionally induced by ZEB1, and promoted the migration and invasion of lung cancer cells. Through the analysis of public ChIRP-seq data, we found that lnc-NR2F1 can modulate the expression of TWIST2, and add-back of TWIST2 restored the migration and invasion of lung cancer cells suppressed by lnc-NR2F1 knockdown. In conclusion, lnc-NR2F1 is induced by a ZEB1-NR2F1 axis and stimulates the migration and invasion of lung cancer cells via transcriptional regulation of TWIST2.

Citation Format: Eun Ju Kim, Young-Ho Ahn. Lnc-NR2F1 upregulated by a ZEB1/NR2F1 axis promotes the migration and invasion of lung cancer cells via TWIST2 regulation [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 1561.
Abstract 1580: Cancer cells promote phenotypic alterations in adjacent hepatocytes to facilitate vessel co-option in colorectal cancer liver metastasis
Authors: Rada Miran , Lazaris Anthoula , Metrakos Peter
Published: June 2022

Vessel co-option is associated with resistance against anti-angiogenic therapy in colorectal cancer liver metastases (CRCLM). Vessel co-opted lesions are characterized by highly motile cancer cells that move toward and along the pre-existing vessels in the surrounding non-malignant tissue and hijack them to gain access to nutrient. In order to access the sinusoidal vessels, the cancer cells must displace the hepatocytes and occupy their space. However, the mechanisms underlying this displacement are unknown. Herein, we examined the involvement of apoptosis, motility, epithelial-mesenchymal transition (EMT) and autophagy pathways in hepatocyte’s displacement by cancer cells. We demonstrated that cancer cells induce the expression of the proteins that associated with upregulation of apoptosis, motility and EMT in adjacent hepatocytes in vitro and in vivo. Accordingly, we observed upregulation of cleaved caspase-3, cleaved poly (ADP-ribose) polymerase-1 (PARP-1) and Actin Related Protein 2/3 (ARP2/3) in adjacent hepatocytes to cancer cells, while we noticed lower expression levels of E-cadherin. Importantly, Runt-related transcription factor 1 (RUNX1) knockdown in the cancer cells has impaired their function towards adjacent hepatocytes in vitro and in vivo. Altogether, our data indicating that cancer cells may exploit various mechanisms to displace the hepatocytes and access to the sinusoidal vessels to establish vessel co-option.

Citation Format: Miran Rada, Anthoula Lazaris, Peter Metrakos. Cancer cells promote phenotypic alterations in adjacent hepatocytes to facilitate vessel co-option in colorectal cancer liver metastasis [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 1580.
Abstract 1582: Chloroquine inhibites Rab11 mediated E-cadherin transport in collective colon cancer cell migration
Authors: Hou Xiang-Ling , Koo Hui Min , Weng Yu-Cheng , Lu Yi-Wen , Winarso Viriya Adhiguna , Li Yu-Lin , Chao Wei-Ting
Published: June 2022

Cell migration is regulated by multiple molecular pathways to change the morphology from epithelial to mesenchymal type and is important in cancer metastasis. In has been demonstrated epithelial mesenchymal transition (EMT) is related to E-cadherin expression level. The loss of E-cadherin expression results in tumor proliferation and metastasis. Recently, studies have been demonstrated the collective cancer cell migration which expresses high E-cadherin is a powerful mechanism of metastasis. Therefore, the role of E-cadherin in cancer metastasis is still not clear. Clathrin-mediated endocytosis (CME) was shown to regulate E-cadherin turnover on the membrane and the function of lysosome is also associated with cell vesicle trafficking, however, the role of lysosome in collective cell migration is not known. This study is investigating the role of lysosome in regulating E-cadherin dynamics through vesicle trafficking in collective colon cell migration. In the experiment, we used colon cancer cells with epithelial morphology and observed the relation of E-cadherin with endocytic protein clathrin, Rab5 and the recycling protein Rab11 in collective migrated cells. In the result, inhibition of lysosome activity by chloroquine treatment has great suppression effect on anchorage independent cell growth and inhibited collective cell migration. We observed that nocodazole treatment inhibited clathrin-mediated endocytosis, which caused E-cadherin to accumulate on the cell membrane. Western blot and confocal microscopy demonstrated chloroquine treatment induced Rab11 expression and accumulated in the cytosol which affected E-cadherin turnover. Our results demonstrated inhibits lysosome activity can suppress colon cancer cell growth and collective migration through lysosome mediated E-cadherin translocation. Chloroquine may be the potential treatment for colon cancer therapy.

keywords: colon cancer, collective cell migration, E-cadherin, vesicle trafficking, lysosome

Citation Format: Xiang-Ling Hou, Hui Min Koo, Yu-Cheng Weng, Yi-Wen Lu, Viriya Adhiguna Winarso, Yu-Lin Li, Wei-Ting Chao. Chloroquine inhibites Rab11 mediated E-cadherin transport in collective colon cancer cell migration [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 1582.
Abstract 1591: Convergence of antigenic diversity and Tex-cell stability fatally constrains immune surveillance in non-small cell lung cancer (TRACERx) and HIV-1 infection (Protocol C)
Authors: Reading James L. , Rosenthal Rachel , Pich Oriol , Gamble Samuel , Ung Seng Anakin , Sahwangarrom Teerapon , Gration Betty , Wong Yien Ning Sophia , Black Lucas , Chain Benny , Hare Jonathan , Quezada Sergio A. , Swanton Charles
Published: June 2022

T cells recognize cognate antigen and elicit effector function to eliminate target cells. In chronic infection and cancer, exhausted T cells with impaired effector function and narrow TCR repertoires must respond to vast and diverse antigenic landscapes, favoring immune escape. Here we test whether T cell exhaustion and antigenic diversity converge to drive fatal immune failure in chronic infection and cancer. We leveraged over 600 primary longitudinal samples from the Protocol C (IAVI) and TRACERx (CRUK) studies to track the natural course of HIV-1 infection and non-small cell lung cancer (NSCLC), respectively, using high parameter flow cytometry, genome sequencing and TCRseq, complemented with focused scRNAseq and antigen specific profiling. In both diseases systemic Tcf1-Tox+Eomes+PD1+ T (Tex) cells track antigen burden and predict adverse outcome, independent of host clinical status. In HIV-1 infection, Tex cells are clonally restricted and rapid Gag RNA sequence divergence accompanies higher viral loads and lower CD4 counts. Similarly, in lung cancer peripheral Tex cells harbor hyper-expanded TCRs, contract after resection and reconstitute at recurrence with strikingly preserved clonal hierarchies, creating a ‘specificity gap’ that could be exploited by subclonal evolution. Commensurate with this model, we discover that a high level of systemic Tex cells coupled with either elevated Gag diversity in HIV-1 infection, or higher mutational or copy number heterogeneity in lung cancer defines patients with the poorest clinical outcome. We propose a unifying model of immune failure in chronic infection and cancer where genomic diversity and T cell exhaustion co-ordinately diminish human immune protection.

Citation Format: James L. Reading, Rachel Rosenthal, Oriol Pich, Samuel Gamble, Seng Anakin Ung, Teerapon Sahwangarrom, Betty Gration, Yien Ning Sophia Wong, Lucas Black, Benny Chain, Jonathan Hare, Sergio A. Quezada, Charles Swanton. Convergence of antigenic diversity and Tex-cell stability fatally constrains immune surveillance in non-small cell lung cancer (TRACERx) and HIV-1 infection (Protocol C) [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 1591.
Abstract 160: IKKα interacts with DARPP-32 to promote lung adenocarcinoma growth
Authors: Alam Sk. Kayum , Wang Li , Zhu Zhu , Hoeppner Luke H.
Published: June 2022

Lung cancer is the leading cause of cancer-related death in the U.S. and worldwide. Non-small cell lung cancer (NSCLC) represents 80-85% of all lung cancer diagnoses, most of which present as advanced disease with poor prognoses. Despite existing treatment options, the overall 5-year survival rate of lung cancer patients is about 17%. Many patients are diagnosed with advanced stage disease that frequently progresses after developing resistance to therapy. Therefore, a better understanding of the molecular mechanisms that control NSCLC progression is necessary to develop new therapies to improve the prognoses of patients. We recently demonstrated that dopamine and cyclic AMP-regulated phosphoprotein, Mr 32000 (DARPP-32), and its truncated splice variant, t-DARPP, promote NSCLC growth using functional studies, orthotopic mouse models, and patient-derived specimens. Specifically, we previously reported that DARPP-32 isoforms promote NSCLC cell migration through activation of non-canonical NF-κB2 signaling by establishing a direct physical interaction with inhibitory kappa B kinase-alpha (IKKα). Therefore, we first sought to determine whether IKKα directly phosphorylates DARPP-32. Indeed, we observed that kinase active IKKα protein phosphorylates DARPP-32 protein at the Thr-34 position using in vitro kinase assays. Given that phosphorylated DARPP-32 acts as a potent inhibitor of protein phosphatase 1 (PP1), we assessed PP1 activity upon transient overexpression of IKKα in DARPP-32-overexpressed NSCLC cells. Our findings suggest phosphorylation of DARPP-32 by IKKα results in decreased PP1-mediated phosphatase activity. This DARPP-32-mediated inhibition of PP1 was not observed in NSCLC cells stably overexpressing a form of DARPP-32 that cannot phosphorylated at the Thr-34 residue (via T34A mutation), suggesting IKKα protein phosphorylates DARPP-32 at the Thr-34 position to stimulate DARPP-32-mediated inhibition of PP1 activity. Correspondingly, we have observed increased phosphorylation of ERK protein, a kinase regulated by PP1, upon pharmacological inhibition of PP1 as well as following overexpression of IKKα protein. Ablation of IKKα via lentiviral transduction reduces NSCLC cell growth in soft-agar colony formation assays. Furthermore, using an orthotopic human NSCLC cell line-derived xenograft mouse model, we have shown that depletion of IKKα reduces tumor growth. Taken together, our findings suggest that IKKα contributes to NSCLC growth by interacting with DARPP-32 to stimulate oncogenic kinase activity through the inhibition of PP1 phosphatase activity.

Citation Format: Sk. Kayum Alam, Li Wang, Zhu Zhu, Luke H. Hoeppner. IKKα interacts with DARPP-32 to promote lung adenocarcinoma growth [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 160.
Abstract 1620: Clonal hematopoiesis of indeterminate potential in the companion dog
Authors: Sebastian Kimberley N. , Gülçin Özer Hatice , Howard Cory , Chadsey Laura , Lozanski Arletta , Doong Tzyy-Jye , Lozanski Gerard , Ma Wenjuan , Kisseberth William C. , Byrd John C. , Harrington Bonnie K.
Published: June 2022

Background: Clonal hematopoiesis of indeterminate potential (CHIP) is a clinical entity of aging humans that is characterized by cancer-associated mutations in white blood cells, without evidence of overt neoplasia. CHIP has been associated with an increased risk of hematologic cancers, cardiovascular disease, and all-cause mortality. We hypothesized that somatic mutations in specific genes associated with human CHIP would be detectable in the blood of aged dogs not known to have hematologic disorders.

Methods: DNA from paired germline and whole blood samples from 93 geriatric canine patients affected by solid cancer were subjected to targeted next generation sequencing. Impact of the variants was predicted using Polymorphism Phenotyping version 2 software (PolyPhen-2, Harvard). Clinical and demographic data were extracted from medical records.

Results: Somatic variants were detected in peripheral blood of four (4.3%) female dogs aged 12-15 years. Affected genes were ASXL1, KIT, SF3B1, TET2, RUNX1, and PPM1D. The variant in PPM1D was a nonsense mutation, while the other five variants were single nucleotide non-synonymous variants in protein coding regions of the genes. Following analysis by PolyPhen-2, the single nucleotide variants in KIT and SF3B1 were predicted to be benign, while the variants in ASXL1, TET2, and RUNX1 were predicted to be damaging. Conclusion: These results support the presence of variants in CHIP-associated genes in geriatric canids at a frequency similar to that observed in people, and the dog represents the first species in which the genetic lesion of CHIP has been documented. Further investigations are needed to confirm the association of this genetic lesion with clinical outcomes.

Citation Format: Kimberley N. Sebastian, Hatice Gülçin Özer, Cory Howard, Laura Chadsey, Arletta Lozanski, Tzyy-Jye Doong, Gerard Lozanski, Wenjuan Ma, William C. Kisseberth, John C. Byrd, Bonnie K. Harrington. Clonal hematopoiesis of indeterminate potential in the companion dog [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 1620.
Abstract 1634: Reduction or loss of Msh2 confers resistance to temozolomide in glioblastoma
Authors: Vallcorba Montserrat Puigdelloses , Buonfiglioli Alice , Piñero Gonzalo , Chen Zhihong , Hambardzumyan Dolores
Published: June 2022

Glioblastoma (GBM) is the most common and aggressive primary brain tumor. The only intervention that has improved the survival rate of GBM patients over the past several decades has been combining temozolomide (TMZ) with radiotherapy (RT), which increased median survival by only ~2.5 months to where it currently stands at ~15 months. Unfortunately, all GBM patients eventually die due to tumor recurrence. Intrinsic or acquired resistance to TMZ is a significant contributing factor to tumor progression, and various mechanisms have been suggested, including deficiencies in DNA mismatch repair (MMR) genes such as MSH2. Utilizing patient GBM samples from the TCGA, we have identified a correlation between low MSH2 expression and shorter patient survival. To test the biological significance of Msh2 in GBM growth and resistance to TMZ and immunotherapy, we have generated immunocompetent genetically engineered mouse models (GEMMs) of GBM with germline or somatic loss of Msh2. To generate tumors with reduced or abrogated Msh2, we utilized GEMMs based on the RCAS/tv-a gene transfer system in combination with mice that exhibited heterozygous and homozygous loss of Msh2. When PDGFB was overexpressed in combination with the silencing of Tp53 to induce tumors, tumor-bearing mice with reduced or deficient Msh2 demonstrated shorter survival time compared to wild-type tumor-bearing mice. In addition, our results indicate Msh2 deficiency increases the malignancy of gliomas. Our data demonstrate that two weeks of TMZ treatment at a clinically relevant dose of 25 mg/kg provides a significant survival advantage in WT-tumor-bearing mice, while no efficacy was observed in tumor-bearing mice with either heterozygous or homozygous loss of Msh2. We are currently evaluating the role of Msh2 in the efficacy of immune checkpoint inhibitors in glioblastoma.

Citation Format: Montserrat Puigdelloses Vallcorba, Alice Buonfiglioli, Gonzalo Piñero, Zhihong Chen, Dolores Hambardzumyan. Reduction or loss of Msh2 confers resistance to temozolomide in 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 1634.
Abstract 1689: Heterogeneity of tumor-initiating cell related signaling revealed by lentiviral signaling reporters in breast cancer patient-derived xenografts
Authors: Landua John , Gong Ping , Lewis Michael T.
Published: June 2022

Classical markers of tumor-initiating cells (TIC), aldehyde dehydrogenase 1 (ALDH1) activity and the cell-surface markers, CD44pos/CD24low/neg, show insufficient cell type specificity to identify TIC definitively, nor do they allow robust enrichment by FACS. As a consequence, analyses of TIC function and gene expression to date are not definitive. Identification and characterization of the TIC subpopulation is critically important in breast cancer since these cells are thought to mediate treatment resistance, as well as local and distant recurrence. This is particularly critical for “triple negative” breast cancer (TNBC), since currently there are no targeted therapies available for this clinical subtype. Lentiviral signaling reporter vectors allow purification of enriched TIC subpopulations thereby facilitating single cell analyses to identify distinct sets of cells with TIC features. We used a collection of fluorescent lentiviral reporters that enable visualization and flow cytometric enrichment of cells undergoing STAT3-, GLI-, WNT-, or OCT4/SOX2-mediated transcriptional activity. Our collection of patient derived xenograft (PDX) models of TNBC were infected with the lentiviral reporters, re-transplanted, and screened for reporter activity upon regrowth. TIC function of reporter-expressing cells relative to negative cells was assayed by mammosphere formation and limiting dilution transplantation. Reporter expressing cells with enriched TIC function were then analyzed by single cell RNAseq for expression of classical markers to identify the subset of cells most likely to represent the TIC subpopulation. Our results indicate that STAT3 and GLI transcriptional activity can serve as uniquely PDX-dependent indicators for TIC function for a subpopulation of cancer cells. Also, multiple potential TIC signaling pathways, including STAT3, GLI, WNT, and OCT4/SOX2, can be active in a single PDX model, but do not necessarily associate with TIC function, indicating TIC diversity in TNBC. These data in conjunction with treatment response and single-cell RNA-seq sheds light on the molecular diversity of TIC populations in TNBC, identifies candidate targets for therapeutic intervention, and will indicate whether TIC diversity is associated with differential response to different chemotherapeutic agents.

Citation Format: John Landua, Ping Gong, Michael T. Lewis. Heterogeneity of tumor-initiating cell related signaling revealed by lentiviral signaling reporters in breast cancer patient-derived xenografts [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 1689.
Abstract 1717: Single-cell RNA sequencing reveals insights into the intra-tumoral heterogeneity and tumor microenvironment of ocular adnexal sebaceous carcinoma
Authors: Gallo Ryan Alexander , Zhang Michelle G. , Tse David T. , Pelaez Daniel , Rong Andrew J.
Published: June 2022

Sebaceous carcinoma (SC) is a malignant neoplasm that predominantly arises from the sebaceous glands of the ocular adnexa. It is the second most common malignant eyelid neoplasm in the United States. No standardized treatment exists, but surgical excision is the mainstay of therapy. Therapeutic advances have been stagnant largely due to a limited understanding of SC tumor biology. We performed single-cell RNA sequencing to profile primary ocular adnexal SC and gain insights into its tumor microenvironment. Ocular adnexal SC has extensive intra-tumoral heterogeneity at the transcriptional level. Dimensionality reduction via principal component analysis was performed to identify major cell types. 8 clusters were detected, including clusters for cancer epithelial cells and various immune cells. Transcriptional programs were related to pathways in cancer, immune response, and antigen processing and processing, among others. We identified unique subpopulations of tumor-infiltrating immune cells, including plasma B cells, GNLY+ natural killer cells, CD8+ T cells, memory T cells, and CD20+ B cells. This complex ecosystem of tumor and immune cells provides new insights into SC biology. Additional patient samples we be analyzed in the immediate future. Follow-up mechanistic studies are needed to clarify the roles of the identified immune cells and how they can be exploited for immunotherapeutic approaches.

Citation Format: Ryan Alexander Gallo, Michelle G. Zhang, David T. Tse, Daniel Pelaez, Andrew J. Rong. Single-cell RNA sequencing reveals insights into the intra-tumoral heterogeneity and tumor microenvironment of ocular adnexal sebaceous carcinoma [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 1717.
Abstract 1719: Investigating intra-tumor heterogeneity using multiplexed immunohistochemistry & deep learning: A new approach to spatially map the tumor microenvironment
Authors: Noureddine Kouther , Gallagher Paul , Guillaud Martial , MacAulay Calum
Published: June 2022

The tumor microenvironment (TME) is a highly complex mixture containing epithelium, stroma and a diverse network of immune cells & the spatial organization of these immune cells within the TME reflects a crucial process in anti-tumor immunity. At present, the usual standard of care for assessing if a patient has cancer, its stage and its likely future biological behaviour is visual examination of one or more stained sections. Although recent advances in multiple immunostaining have enabled characterization of several parameters on a single tissue section. For a higher dimensional chromogen based methodology, we have developed a multiplexed IHC (mIHC) procedure combining multiple labels per round with several sequential rounds, enabling analysis of complex immune cell populations on a single slide through consecutive cycles of staining, destaining & hyperspectral imaging. By integrating serial imaging, sequential labeling & image registration, we are able to spatially map the TME. The process presented is using absorption microscopy, enabling these images to be done in a reasonable time frame.

Robust, accurate, segmentation of cell nuclei for overlapping nuclei is one of the most significant unsolved issues in digital pathology. Using the counterstain concentration images from our mIHC methodology, we have trained a deep learning segmentation method to accurately segment individual cell nuclei within overlapping clusters of nuclei. By combining a multiplexed IHC technique which enables the detection of multiple markers on a single slide with deep learning segmentation methods to segment every individual cell nuclei in tissue sections with an accuracy comparable to human annotation. We can analyze the cell-cell interactions between immune and tumour cells, enhancing our ability to perform molecularly based single cell analysis of multiple cell types simultaneously within the tissue. These two techniques joined can be scaled up to the entire tissue section level, improving our understanding of the biological aggressiveness of specific cancers & enabling an accurate spatial cell level representation of the tissue.

Citation Format: Kouther Noureddine, Paul Gallagher, Martial Guillaud, Calum MacAulay. Investigating intra-tumor heterogeneity using multiplexed immunohistochemistry & deep learning: A new approach to spatially map the 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 1719.
Abstract 1740: Determining the intracellular fate of ligand-conjugated therapeutics using nanogold labeling
Authors: Sohal Ikjot S. , Abdelaal Ahmed M. , Kasinski Andrea L.
Published: June 2022

Recent developments in ligand-conjugated RNAi therapeutics have led to improved stability of RNAi molecules, reducing effective therapeutic dose, and avoiding delivery vehicle-associated toxicity. However, as our group has shown in previous studies with FolamiR (Folate-conjugated miR-34a), endosomal entrapment of the therapeutic molecule is a rate-limiting factor in RNAi activity. To improve endosomal release of ligand-conjugates, several approaches are being explored such as incorporation of endosomal escape agents (nigericin), chemical modification to enhance stability of miRNA/siRNA within the acidic compartments, and modification of ligand-receptor interaction to enable adequate release from the receptor upon internalization. Current fluorescent microscopy techniques are only able to approximately estimate the cellular fate of FolamiR without providing enough resolution to determine the endosomal stage, receptor disengagement and other dynamics involved in the endosomal entrapment/release of FolamiR. To overcome this, we developed Folate-conjugated nanogold construct (FolaGold) that mimics FolamiR molecule. The nanogold in FolaGold is used as a proxy for miR-34a and electron microscopy can be performed to pinpoint the cellular fate of nanogold including receptor disengagement process, the endosomal stage as well as cytosolic enrichment at the nanometer resolution. In this study, FolaGold is validated to bind to folate receptor in a competition assay and is confirmed to be present in specific endosomal compartments in MB231 cells, unlike unconjugated nanogold. We further plan to use FolaGold to determine changes in intracellular fate of nanogold upon incorporation of endosomal escape agents (nigericin) and modification of ligand-receptor interaction by using a lower affinity binding ligand that facilitates ligand-receptor disengagement following internalization. While we are using nanogold labeling to determine the intracellular fate of ligand-conjugated RNAi therapeutics, the labeling can also help improve our understanding of endosomal release dynamics and activity of other ligand-conjugate based therapeutics.

Citation Format: Ikjot S. Sohal, Ahmed M. Abdelaal, Andrea L. Kasinski. Determining the intracellular fate of ligand-conjugated therapeutics using nanogold labeling [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 1740.
Abstract 1752: Switchable binding of TecApta to Atezolizumab provides the possibility of enhancing the therapeutic effects of this immune checkpoint inhibitor
Authors: Atefi Mohammad Sadegh , Takahashi Sola , Sich David , Grondin Jessica , Doueiri Rami , Bourgeois Lindsay , Olsem Gary , Futamura Akiko , Yang Julie , Oliva Joan , Elshimali John , Pereira Carlos
Published: June 2022

One of the main goals of cancer therapeutics is to develop smart molecules that can deliver drugs or immunogens to cancer cells within the tumor, while avoiding the toxicity to normal cells. Lately, immune checkpoint inhibitors have shown significant effects in the field of cancer treatment. Atezolizumab is an immune check point inhibitor specific to PD-L1. In order to potentiate anti-tumor aspects of Atezolizumab, we have developed TecApta, a switchable single strand DNA oligonucleotide aptamer that specifically binds to this antibody with a high affinity and specificity. However, TecApta is detached from Atezolizumab upon binding of this antibody to its target PD-L1. This switchable aspect of TecApta makes it a suitable drug carrier for targeting cancer cells within the tumor microenvironment. We have developed modified versions of TecApta which include either the chemotherapy drug, gemcitabine, or the immune activator, TLR9A. Based on our in vitro data, both versions are biologically active. The gemcitabine sensitive cell line MDA-MB-231/Luc exhibits similar IC50s (20nM) for both gemcitabine-linked-TecApta (GMZ-TecApta) and the free unbound gemcitabine. Moreover, the same concentrations of TLR9A-linked-TecApta (TLR9A-TecApta) and the free TLR9A show similar effects in inducing TLR9 receptors in a cell-based assay. These results suggest that possibly GMZ-TecApta and TLR9A-TecApta can be carried by Atezolizumab and released within the tumor microenvironment, where they may induce apoptosis or aggravate an immune reaction, respectively. Animal studies will be conducted in the future to investigate the possibility of such favorable outcomes.

Citation Format: Mohammad Sadegh Atefi, Sola Takahashi, David Sich, Jessica Grondin, Rami Doueiri, Lindsay Bourgeois, Gary Olsem, Akiko Futamura, Julie Yang, Joan Oliva, John Elshimali, Carlos Pereira. Switchable binding of TecApta to Atezolizumab provides the possibility of enhancing the therapeutic effects of this immune checkpoint inhibitor [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 1752.
Abstract 176: Development of PDX-derived spheroids using LifeGel, an innovative 3D cell culture technology
Authors: Pietrzyk Agnieszka , Indersie Emilie , Deas Olivier , Judde Jean-Gabriel , Krzykawski Marcin , Cairo Stefano
Published: June 2022

One of the biggest challenges in preclinical oncology is the lack of models faithfully recapitulating the physiological and pathophysiological features of the native tumor. PDXs (Patient-Derived Xenografts), developed in immune-compromised mice, have proven their relevance in the study of aberrations leading to the development and progression of cancer, to the mechanisms linked to tumor resistance and to the identification of novel therapies. Despite being a fundamental step to generate new knowledge on cancer, the use of PDXs shows some technical disadvantages such as long engraftment time and/or low growth rate, thus raising economic and ethical concerns. Huge efforts are being done to develop cell-based alternatives systems reproducing the tumor complexity combined with suitable methods. To this aim, we have developed 50+ PDX-derived cell lines (PDXDCs) from our proprietary 200+ PDX collection. These lines are currently used as two-dimensional (2D) cultures to perform drug screening in an affordable, timewise, and cost-effective way. While well designed, drug-tailored 2D screenings can predict drug behavior in vivo, they present limitations as they are unable to reproduce cell-cell and cell-extracellular matrix interactions, two important parameters that contribute to the regulation of drug accessibility and oxygen diffusion in vivo. To encompass those limitations and give access to a wider range of experimental methods we have generated three-dimensional (3D) cell culture models by taking advantage of LifeGel technology, consisting of protein-based hydrogels that modulate matrix density and stiffness to mimic the organ of origin-specific extracellular environment. Thanks to its versatility we successfully developed spheroid cultures from PDXDCs from various indications, including breast cancer cells from different histological subtypes. Tumor cells plated on LifeGel adopted 3D spheroid-like structures in a few days. These structures showed heterogeneous morphology and size depending on the tumor type and the hydrogel composition. As spheroids can be passaged in vitro by digestion, we could easily expand them and perform drug screening that will be presented and compared to 2D and in vivo results. The aim of this project is to develop a collection of PDX-derived spheroids recapitulating PDXs characteristics. This collection will give the opportunity to perform high-content drugs screening to select the best candidate drugs to be retained for further preclinical in vivo studies.

Citation Format: Agnieszka Pietrzyk, Emilie Indersie, Olivier Deas, Jean-Gabriel Judde, Marcin Krzykawski, Stefano Cairo. Development of PDX-derived spheroids using LifeGel, an innovative 3D cell culture technology [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 176.
Abstract 1786: SM-88, D/L-alpha-metyrosine, is a novel anti-cancer agent in estrogen receptor positive breast cancer
Authors: Demas Diane M. , Collins Julie , Shajahan-Haq Ayesha N.
Published: June 2022

Breast cancer is the second most common cause of death from cancer in women in the United States after lung cancer. Estrogen receptor positive (ER+) breast cancer comprises 70% of all breast cancer cases. Antiestrogens and CDK4/6 inhibitors are now part of standard of care therapies for advanced and metastatic ER+ breast tumors. Unfortunately, resistance to these therapies is prevalent and metastatic breast cancer remains an incurable disease. Since drug resistant cancer cells often have deregulated metabolic pathways, we tested the efficacy of a novel dysfunctional tyrosine, SM-88 (D,L-alpha-metyrosine; racemetyrosine), alone or in combination with sub-toxic doses of conditioning agents, methoxsalen, phenytoin, and sirolimus (MPS) in resistant cell models of ER+ breast cancer. In normal cells, tyrosine, a non-essential amino acid, is made from phenylalanine and not readily taken up by cells. Therefore, it is hypothesized that SM-88 uptake will be increased in cancer cells compared to normal cells. To evaluate whether SM-88 is a potential anti-cancer agent for drug resistant ER+ tumors, we used antiestrogens and CDK4/6 inhibitors sensitive (parental MCF7 and T47D cell lines) or resistant (MCF7-R and T47D-R) cells in this study. We show that cell proliferation was inhibited in both sensitive and resistant ER+ breast cancer cells at 72 h, however, the IC50 of SM-88 as a monotherapy was higher in resistant cells compared with sensitive cells, 5mM versus 2 mM, respectively. Combination of SM-88 and MPS had an additive effect and lowered the IC50 in both resistant and sensitive cells, 2mM versus 1mM, respectively. Both LAT1 and CD98, known transporters of tyrosine, were expressed in sensitive and resistant cells. Furthermore, SM-88 as a single agent or in combination with MPS resulted in the induction of incomplete autophagy in both sensitive and resistant cells. Apoptosis and DNA damage was noted in p53-wildtype MCF7 and MCF7-R but not in p53-mutant T47D and T47D-R cells with SM-88 monotherapy or in combination with MPS. Ongoing work is focused on further defining the biochemical pathways in drug sensitive and resistant cells that will further support the current clinical use of SM-88 in advanced ER+ breast cancer (ClinicalTrials.gov Identifier: NCT04720664). Collectively, we show that SM-88 is potentially a novel anti-cancer agent in drug resistant ER+ breast cancer.

Citation Format: Diane M. Demas, Julie Collins, Ayesha N. Shajahan-Haq. SM-88, D/L-alpha-metyrosine, is a novel anti-cancer agent in estrogen receptor positive breast 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 1786.
Abstract 184: High-throughput chemotherapeutic drug screening of tumor spheroids with individual spheroid results using image cytometry
Authors: Bell Jordan , Mukundan Shilpaa , Teryek Matthew , Lin Bo , Parekkadan Biju , Chan Leo
Published: June 2022

Three-dimensional cancer models have gained popularity for in vitro studies of chemotherapeutic compounds by providing a more physiologically relevant analog of gas, nutrient, and drug diffusion throughout the tumor microenvironment. Some 3D assays are performed to study individual spheroids over time, where a majority of these assays rely on maintaining a single spheroid in each well of a 96-well round-bottom ultra-low attachment plate, limiting the number of spheroids in a study. Other assays may gather population-level data from large ensembles of spheroids grown together, but the information about individual differences amongst the spheroids is lost. Important kinetic information may also be lost for destructive endpoint assays such as MTS or MTT. Here, we describe the development of a 3D image cytometry assay that is capable of generating kinetic data for thousands of breast cancer spheroids at the individual level. T47D spheroids are grown and maintained in a 24-well Aggrewell࣪400 plate and imaged using the Celigo image cytometer. Each well contains more than 1000 subwells that both aid in spheroid formation and constrain each spheroid to a specific location. Using the spheroid location data, we are able to track and monitor the growth of each spheroid over 7 days. Furthermore, we investigate the dose-dependent effects on spheroid viability of 6 anti-cancer drugs (Doxorubicin, Everolimus, Gemcitabine, Metformin, Paclitaxel and Tamoxifen) using calcein AM and propidium iodide (PI). To validate the viability measurement results, we utilize the CellTiter96® MTS assay as an orthogonal method to compare the dose-dependent trends using both the calcein AM and PI fluorescence intensities as well as the spheroid sizes. This work may lay a foundation for the investigation of other spheroids, organoids, or tissue samples, significantly increasing the number of spheroids analyzed per condition, improving the statistical analysis, and adding more parameters to further analyze the spheroids. These improvements may be especially helpful for spheroids grown from patient-derived or otherwise heterogeneous cell populations

Citation Format: Jordan Bell, Shilpaa Mukundan, Matthew Teryek, Bo Lin, Biju Parekkadan, Leo Chan. High-throughput chemotherapeutic drug screening of tumor spheroids with individual spheroid results using image cytometry [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 184.
Abstract 189: Evaluating the influence of Lactobacillus rhamnosus-derived extracellular vesicles on calcium deposition in early breast cancer
Authors: Vuong Ngoc Bao , Alomia Melany , Alruwaili Intisar , Alsaab Fahad , Erickson James L. , Zhou Weidong , Luchini Alessandra , Van Hoek Monique , Espina Virginia , Hoefer Richard A. , Liotta Lance
Published: June 2022

Microcalcifications are an important mammographic feature of ductal carcinoma in situ (DCIS) and are detected in 85-90% of abnormal mammograms. Calcium transport and calcium channels regulate microcalcifications and are essential for breast cancer proliferation. The breast duct intraductal lumen, which is the origin of ductal carcinomas, has been shown to be a non-sterile environment. We hypothesize that the intraductal microbiome influences calcium handling and mineral deposition in breast cancer. Bacteria have been known to enhance the formation of mineral deposits, including calcium salts, in various non mammalian microenvironments via secreted factors or extracellular vesicle mediated nucleation. We performed a clinical study on a cohort of 150 women who received a suspicious mammogram and were subjected to a follow up biopsy, and we identified differences in calcium handling microbiota between patients who harbored a malignant disease with respect to benign controls. To mechanistically study the role of microbiota in breast cancer calcium handling, we developed a 3D spheroid model of ductal carcinoma in situ using the BT-474 cell line. When exposed to high concentrations of calcium in the culture medium (from 1.75 mM to 2.05 mM), spheroids developed calcium mineral deposits in a calcium dose dependent manner; deposits were identified in internal regions of the spheroid, which correspond to hypoxic areas of DCIS in vivo. Extracellular calcium deposition correlated with cell survival in face of a toxic calcium level. Spheroids were cultured under a continuous flow of Lactobacillus rhamnosus LMS2-1-derived EVs over the course of two weeks. EVs were isolated by sequential ultracentrifugation; the 100K fraction was retained and characterized in terms of size, concentration, and protein content. EV treatment induced increased calcium export and mineral deposition in a dose dependent manner. Our findings support the hypothesis that L. rhamnosus-derived extracellular vesicles influence calcium regulation and mineral deposition in the hypoxic breast cancer milieu. More studies will be undertaken in the future to unveil molecular mechanisms that underlie this interaction, both at the cancer cell and the microbiota level.

Citation Format: Ngoc Bao Vuong, Melany Alomia, Intisar Alruwaili, Fahad Alsaab, James L. Erickson, Weidong Zhou, Alessandra Luchini, Monique Van Hoek, Virginia Espina, Richard A. Hoefer, Lance Liotta. Evaluating the influence of Lactobacillus rhamnosus-derived extracellular vesicles on calcium deposition in early breast 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 189.
Abstract 1892: Expression of bacterial heme-sequestering proteins (HeSPs) in Pichia pastoris for effective treatment of lung cancer
Authors: Salamat Narges , Ashrafi Adnin , Wang Tianyuan , Akter Zakia , Gong Hanling , Zhang Li
Published: June 2022

Heme is a central biosynthetic molecule with important implications in diverse molecular and cellular processes, including oxygen utilization and metabolism. Heme carries out a vital role in mitochondrial energy generation through oxidative phosphorylation (OXPHOS) by acting as a cofactor or prosthetic group for complexes II, II, and IV in the electron transport chain. Lung cancer is the leading cause of cancer related death in the US. Our lab has previously shown that non-small cell lung cancer (NSCLC) cells exhibit an elevated heme flux characterized by elevated heme synthesis and import. This increased heme flux correlates with an enhanced oxygen consumption and ATP production. To target this elevated heme flux, we have engineered recombinant bacterial HasA hemophores as heme-sequestering proteins (HeSPs). HeSPs bind to and sequester heme with an extremely high (pM) affinity, thereby disrupting this elevated heme flux. HeSPs display potent anti-tumor activity in mouse models of NSCLC and triple-negative breast cancer (TNBC). Here, we utilized the Pichia pastoris expression platform to achieve high-yield expression. P. pastoris is a methylotrophic yeast that has minimal endogenous protein secretion and potentially high-yields for heterologous protein expression, due to a tightly regulated alcohol oxidase gene (AOX1). The P. pastoris expression platform is generally easier to handle and requires simpler fermentation conditions than mammalian cell cultures, making it an ideal candidate for expression of recombinant bacterial proteins without endotoxin contamination.We utilized a secretion signal to direct secretion of HeSP2 into the culture medium to allow for easier protein purification. Purification was performed through ion exchange chromatography. We utilized native PAGE and heme-binding spectrum to verify that these yeast-expressed HeSPs adopt a similar conformation to those expressed in bacteria and maintain their heme-binding activity. HeSPs expressed through this P. pastoris expression system demonstrated remarkable anti-cancer efficacy in proliferation assays on NSCLC cells and SCLC (small cell lung cancer) cells. Experiments are underway to test their anti-tumor efficacy in mouse models. These experiments may suggest a novel approach to generate effective anti-cancer agents.

Citation Format: Narges Salamat, Adnin Ashrafi, Tianyuan Wang, Zakia Akter, Hanling Gong, Li Zhang. Expression of bacterial heme-sequestering proteins (HeSPs) in Pichia pastoris for effective treatment of lung 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 1892.