bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 155: An endosomal type Igamma PIP 5-kinase controls endosome maturation, lysosome function, and autophagy by modulating Rab7a
Authors: Sun Ming , Luong Gary , Plastikwala Faiz , Sun Yue
Published: June 2022

Endosomes are major protein-sorting stations in cells. Defects in endosomal sorting are implicated in various human pathologies, such as cancer and neurodegenerative disorders. The small GTPase Ras-related protein Rab-7a (Rab7a) serves as a key organizer of the endosomal-lysosomal system. However, molecular mechanisms controlling Rab7a activation levels and subcellular translocation are still poorly defined. Here, we demonstrate that type Igamma phosphatidylinositol phosphate 5-kinase i5 (PIPKIgammai5), an endosome-localized enzyme that produces phosphatidylinositol 4,5-bisphosphate (PI4,5P2), directly interacts with Rab7a and plays critical roles in the control of endosomal-lysosomal system. The loss of PIPKIgammai5 blocks Rab7a recruitment to early endosomes, which prevents the maturation of early to late endosomes. In this way, PIPKIgammai5 is required for Epidermal Growth Factor Receptor (EGFR) sorting from endosomes to lysosomes, which controls the down-regulation of EGFR signaling. Furthermore, PIPKIgammai5 loss disturbs retromer complex connection with Rab7a, which blocks the retrograde sorting of Cation-independent Mannose 6-Phosphate Receptor (CI-MPR) from late endosomes. This leads to the decreased sorting of hydrolases to lysosomes. Thus, loss of PIPKIgammai5 causes lysosome dysfunction and reduces the autophagic degradation. By modulating retromer-Rab7a connection, PIPKIgammai5 is also required for the recruitment of the GTPase-activating protein (GAP) TBC1 domain family member 5 (TBC1D5) to late endosomes, which controls the conversion of Rab7a from the active state to the inactive state. Altogether, PIPKIgammai5 is critical for the modulation of Rab7a activity, localization, and function in the endosomal-lysosomal system.

Citation Format: Ming Sun, Gary Luong, Faiz Plastikwala, Yue Sun. An endosomal type Igamma PIP 5-kinase controls endosome maturation, lysosome function, and autophagy by modulating Rab7a [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 155.
Abstract 1558: Drug repurposing strategies for pancreatic cancer management
Authors: Gaikwad Shreyas R. , Srivastava Sanjay K.
Published: June 2022

Pancreatic ductal adenocarcinoma (PDAC) is the currently the third leading cause of cancer related deaths and is projected to be the second leading cause by 2030. The difficulty in diagnosis and aggressiveness of the disease makes it a difficult-to-treat cancer. Currently, treatment of PDAC mainly involves use of standard chemotherapeutic combinations. Although effective, the current options do not always provide a benefit and patients develop resistance over time. Additionally, the adverse effect profile of standard regimen is a concern. Thus, novel agents which offer better clinical and survival response are urgently required. Drug repurposing is an effective strategy to overcome the limitations in current treatment options. Through drug repurposing, we identified the anti-cancer effects of a compound MBO in human PDAC cell lines AsPC-1, MiaPaCa-2, BXPC3, Panc-1 and SUIT-2. Further, we observed the ability of MBO to suppress colony formation in PDAC cell lines AsPC-1, MiaPaCa-2 and BXPC3. Additionally, MBO induced cell death by apoptosis in PDAC cell lines which was confirmed by Annexin V/FITC analysis using flow cytometry. MBO significantly suppressed tumor growth in a subcutaneous model of PDAC. Studies are currently ongoing to explore the mechanism of action as well as effect of MBO in orthotopic PDAC models.

Citation Format: Shreyas R. Gaikwad, Sanjay K. Srivastava. Drug repurposing strategies for pancreatic cancer management [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 1558.
Abstract 1566: Influence of age on fibroblast derived extracellular vesicles and their role in melanoma progression
Authors: Hueser Laura , Chhabra Yash , Gololobova Olesia , Fane Mitchell , Witwer Kenneth , Weeraratna Ashani
Published: June 2022

Age is an important prognostic factor in cutaneous melanoma, which commonly arises in the elderly. Melanoma progression is the concerted outcome of cellular and molecular changes that occur not just within the tumor cells but also within the milieu of the stromal cells (fibroblasts, immune cells, adipocytes), collectively referred to as the tumor microenvironment (TME). Moreover, TME-secreted growth factors, cytokines, and extracellular matrix (ECM) are vital to tumor progression and therapy resistance. Aging causes remodeling of the TME that becomes conducive to melanoma progression. In addition, extracellular vesicles (EVs) have been shown to transport lipids, mRNAs and functional proteins between different cells in vitro and across tissues in vivo. EVs have also been shown to play an important role in tumor progression in different cancer types, and aging can change their composition as well as their cargo. We performed proteomics on EVs released by young and aged dermal fibroblasts, the predominant stromal cell population of melanoma TME, to investigate how aging of the cells changes the EVs released by them. Moreover, we performed functional assays to investigate what influence this will have on the tumor progression. Our study shows that EVs secreted by dermal fibroblasts change in their composition as well as their cargo adding another dimension to age-associated increase in melanoma progression, and therapy resistance.

Citation Format: Laura Hueser, Yash Chhabra, Olesia Gololobova, Mitchell Fane, Kenneth Witwer, Ashani Weeraratna. Influence of age on fibroblast derived extracellular vesicles and their role in melanoma progression [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 1566.
Abstract 1583: The interaction of stromal components and tumor cells dictates the tumor progression and clinical outcomes in stomach cancer
Authors: Lee Seungho , Kim Taemin , Cheong Jaeho , Kim Hyongbum , Lee Sunghwan
Published: June 2022

A subset of gastric cancers (GC) show activated epithelial-to-mesenchymal transformation (EMT), but it is of debate as to the origins of the EMT signals (i.e., from tumor cells or microenvironments) and how they are associated with clinical outcomes. In this study, we observed that mesenchymal GCs comprising 30-40% of datasets showed elevated level of infiltrating stromal cells and worse clinical outcomes. The analysis of xenograft sequencing revealed that the transcriptional signals representing EMT mainly come from stromal cells instead of tumor cells. In addition, no evidence of tumor-originating, EMT-driven stromal cells was found in single cell RNA sequencing data. Interestingly, expression of EMT signature genes in primary tumor was highly correlated with that of mouse genes. Microenvironment profiling identified a specific T cell subtypes associated with mesenchymal GCs such as T cell gamma-delta. Finally, deconvolution-based tumor, immune and stromal cells in mesenchymal GCs showed the YAP1 activation, representing the consequences of interactions of stromal component and cancer as well as defining the intrinsic and extrinsic hallmarks of mesenchymal GCs.

Citation Format: Seungho Lee, Taemin Kim, Jaeho Cheong, Hyongbum Kim, Sunghwan Lee. The interaction of stromal components and tumor cells dictates the tumor progression and clinical outcomes in stomach 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 1583.
Abstract 1592: Pulsatile Myc expression as a source of Myc heterogeneity in cancer cells
Authors: Liu Chad , Gascoigne Karen
Published: June 2022

Myc is the most commonly amplified oncogene, and is known to play critical roles in cancer establishment and maintenance. Myc expression heterogeneity has also been linked to cancer drug resistance; however, the source of this heterogeneity remains poorly characterized. Even in isogenic cancer cell lines, we found that Myc is one of the most heterogeneously expressed transcription factors at the single-cell level. This heterogeneity is independent of cell cycle progression, local cell density, and p53/p21 signaling. To elucidate the source and duration of Myc heterogeneity, we endogenously tagged Myc with a fluorescent protein and used long-term live-cell imaging techniques to monitor Myc expression over time at single-cell resolution. Surprisingly, we found that Myc expression is pulsatile, explaining why Myc expression appears heterogeneous at a given point in time. Furthermore, we found that Myc dynamics are heterogeneous, and the dynamics are not always heritable. Additionally, Myc pulse amplitude is correlated with the duration of G1 and the overall cell cycle, suggesting that the pulses may be a mechanism for escaping many chemotherapy agents. Finally, we show that Myc pulses are primarily transcription-driven and controlled by at least one negative feedback loop. Our findings demonstrate for the first time that Myc expression is pulsatile, and provide insights into Myc regulation in cancer.

Citation Format: Chad Liu, Karen Gascoigne. Pulsatile Myc expression as a source of Myc heterogeneity in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1592.
Abstract 1597: Characterization of genomic diversity in high grade serous ovarian and triple negative breast cancer at single cell resolution
Authors: Williams Marc J. , Funnell Tyler , O'Flanagan Ciara H. , McPherson Andrew , Aparicio Samuel , Shah Sohrab P.
Published: June 2022

Copy number alterations and structural variants are associated with disease progression, therapeutic response, and metastasis in human cancers, yet the extent and mechanisms driving continued genomic instability remain poorly understood. We generated more than 20,000 single-cell whole genomes from 25 high-grade serous ovarian and triple-negative breast cancers across a range of mutational subtypes. Through the development of new computational methods, we resolved alterations into cancer haplotypes, providing fine-grained resolution of genomic events in single-cells and exposing extensive cell-to-cell genomic variation. We identified 3 key processes that contribute to this diversity. Firstly, we observed that parallel copy number events - whereby different cancer cells harbor chromosomal aberrations with identical total copy number but different combinations of maternal and paternal alleles - were present in > 80% of the tumors. This is consistent with extensive convergent evolution in these tumors. Secondly, we identified considerable cell-to-cell variability in the amplitude of high-level amplifications, often impacting oncogenes. Single-cell transcriptomes and immunohistochemistry from the same samples showed that this genomic diversity led to phenotypic differences between cells. Finally, we observed copy number break-point variability consistent with progressive genomic diversification, which we termed serriform structural variation (SSV). Both SSV’s and variable high-level amplifications were enriched in foldback inversion (FBI) bearing tumors relative to homologous recombination deficient tumors, highlighting that FBI tumors have greater levels of structural instability. Our study sheds new light on the evolution of genomically unstable tumors with implications for cancer evolution, therapeutic targeting, and patient stratification.

Citation Format: Marc J. Williams, Tyler Funnell, Ciara H. O'Flanagan, Andrew McPherson, Samuel Aparicio, Sohrab P. Shah. Characterization of genomic diversity in high grade serous ovarian and triple negative breast cancer at single cell resolution [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 1597.
Abstract 1602: Lineage plasticity enables low ER luminal tumors to evolve and gain basal-like traits
Authors: Mohamed Gadisti Aisha Nurulhijjah Binti , Ognjenovic Nevena B. , Mahmood Sundis , Lee Sarah Min Kyung , Christensen Brock C. , Muller Kristen E. , Pattabiraman Diwakar R.
Published: June 2022

Stratifying breast cancer into specific molecular or histological subtypes aids in therapeutic decision-making and predicting outcomes, however, these subtypes may not be as distinct as previously thought. Patients with luminal-like, Estrogen Receptor (ER)-expressing tumors have better prognosis than patients with more aggressive, triple-negative or basal-like tumors. There is, however, a subset of luminal-like tumors that express lower levels of ER, which exhibit more basal-like features. Previous studies have suggested that triple negative, basal-like tumors may arise from a luminal cell-of-origin, but there are no definitive studies that identify the cell-of-origin of these low ER tumors.

Analysis of 2208 invasive breast carcinomas from 2012-2020 revealed that 2% of tumors have low ER expression (less than 10% ER positive cells), which are mostly high-grade carcinomas and exhibit basal-like features. TCGA analysis revealed that tumors with lower ER expression (lowest quartile of ER expression) expressed higher basal signature genes as compared to tumors with higher levels of ER expression. This variation within the ER+ subtype and the emergence of basal-like characteristics within low ER tumors suggest that some luminal tumors may evolve into a more basal-like or triple-negative subtype.

The luminal mouse mammary tumor, MMTV-PyMT, was used to model low ER human tumors and, similar to the patient tumor samples, basal-like tumor cells were also found within these tumors. Lineage tracing using tissue-specific and inducible Cre recombinase-based labelling was performed to elucidate the lineage-of-origin of these basal-like cells, revealing that these basal-like cells were derived from normal luminal epithelial cells, not basal cells.

Our study uncovers the existence of luminal-basal plasticity within tumors of a low ER subtype that enables these cells to transition into a more basal-like state. Understanding the factors that enable this plasticity to occur may reveal opportunities to curb the evolution of more aggressive traits, potentially improving the way breast cancer is currently managed and treated.

Citation Format: Gadisti Aisha Nurulhijjah Binti Mohamed, Nevena B. Ognjenovic, Sundis Mahmood, Sarah Min Kyung Lee, Brock C. Christensen, Kristen E. Muller, Diwakar R. Pattabiraman. Lineage plasticity enables low ER luminal tumors to evolve and gain basal-like traits [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 1602.
Abstract 1618: OP-1250 prevents tumor spread in a model of metastatic mutant ERα+ breast cancer
Authors: Laine Muriel , Greene Marianne E. , Leng Tiffany , Li Sophia , Palanisamy Gopinath S. , Harmon Cyrus L. , Hodges-Gallagher Leslie , Kushner Peter J. , Greene Geoffrey L.
Published: June 2022

Estrogen receptor positive (ER+) breast cancers represent about 70-75% of all breast cancer. In general, these cancers are effectively treated with adjuvant endocrine therapies, with or without the addition of CDK4/6 inhibitors. However, many patients develop resistance and eventually progress to metastatic disease (MBC). Acquired mutations in ESR1, which encodes estrogen receptor alpha (ERα), contribute to 20-40% of endocrine therapy resistant MBC. In patients, breast cancer metastasis occurs in the liver, lungs, brain and bone. OP-1250 is a novel compound that is a complete ERα antagonist (CERAN). Previous studies have shown that OP-1250 effectively blocks both the AF-1 and AF-2 activation functions of ERα. In the current study, we used a mutant ERα MBC endocrine therapy resistant xenograft model to examine the efficacy of OP-1250 in a metastatic context. MCF7 cells engineered to express one of the most common and aggressive ERα mutations, Y537S, were labeled with luciferase and injected via the nipple (mammary intraductal MIND model) into NSG mice. Mice were treated with 3 and 10 mg/kg of OP-1250 alone or in combination with palbociclib at 70mg/kg and tumor growth was monitored via a Xenogen IVIS imager. At study end point, mice were sacrificed and excised organs were imaged ex-vivo and further processed for immunohistology analysis. OP-1250 at both doses inhibited primary tumor growth as well as metastasis to the lung, liver, brain and bone, with greater effect at 10 mg/kg, and was significantly more effective than Faslodex. When combined with palbociclib at 70 mg/kg, an additional significant decrease in metastasis was observed, compared to OP-1250 or palbociclib alone. Our results also suggest that the efficacy seen in the combination study was driven primarily by OP-1250. In total, these results demonstrate the potential use of OP-1250, both alone and in combination with a CDK4/6 inhibitor, to inhibit tumor growth and metastasis in a model of aggressive mutant ER+ MBC.

Citation Format: Muriel Laine, Marianne E. Greene, Tiffany Leng, Sophia Li, Gopinath S. Palanisamy, Cyrus L. Harmon, Leslie Hodges-Gallagher, Peter J. Kushner, Geoffrey L. Greene. OP-1250 prevents tumor spread in a model of metastatic mutant ERα+ 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 1618.
Abstract 1621: Comparison and consequences of different implantation techniques on the orthotopic growth of syngeneic Hepa1-6 liver cancer cells
Authors: Obodozie Cynthia , Moor Sandra , Bijelic Gojko , Malaisé Muriel , Metzger Philipp , Weber Holger
Published: June 2022

Checkpoint inhibitor treatment has become a common therapy of various cancer types. Still, there is a need for well-characterized preclinical mouse models, as clinical data indicates that patients only partially respond to immune modulatory regiments. When compared to the classical subcutaneous or subQperior࣪ (implantation into the mammary fat pad) syngeneic mouse models, orthotopic models are considered more predictive, since implantation of tumor cells into the organ of origin allows organotypical interaction between tumor cells and the surrounding stroma, including immune cells.

Up until now, our standard procedure for an orthotopic Hepa1-6 liver model is the injection of Hepa1-6 cells into the spleen with the subsequent migration of the injected Hepa1 6 cells into the liver for five minutes via Vena lienalis. A major drawback of this method for a syngeneic model in immune-competent mice is the following resection of the spleen, a prominent secondary lymphoid organ. Therefore, we tested two other implantation methods: the direct injection of Hepa1-6 cells into a liver lobe using Matrigel࣪ and the injection of Hepa1-6 cells into the portal vein. In contrast to the standard procedure, both new test methods have in common an excessive bleeding potential which must be prevented.

As the Hepa1-6 cells are transduced with luciferase, their growth can be monitored in vivo by bioluminescence imaging and the growth characteristics will be compared. In addition to the growth, the composition of the immune populations will be analyzed staining with the all-in-one flow cytometry panel which permits the differentiation of the main immune cell populations in the tumor, such as T cells (CD4+, CD8+, Treg), B and NK cells, macrophages (M1/M2), MDSCs (granulocytic and monocytic), and dendritic cells. The data will help to select the most suitable method for testing new drug candidates in an orthotopic liver tumor environment.

Citation Format: Cynthia Obodozie, Sandra Moor, Gojko Bijelic, Muriel Malaisé, Philipp Metzger, Holger Weber. Comparison and consequences of different implantation techniques on the orthotopic growth of syngeneic Hepa1-6 liver cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1621.
Abstract 1622: Signaling of cyclin-dependent kinase 12 (CDK12) in prostate cancer cell lines
Authors: Chalfant Victor , Riveros Carlos , Shukla Sanjeev , Osumi Teruko , Balaji K
Published: June 2022

Cyclin-dependent kinase 12 (CDK12) belongs to the cyclin-dependent kinase (CDK) family of serine/threonine protein kinases. In contrast to CDKs which promote cell cycle progression, CDK12 is a transcriptional regulator of various cellular functions, most importantly cellular response to DNA damage and stress. Genomic alterations in CDK12 have been detected in up to 7% of patients with metastatic castration-resistant prostate cancer (CRPC). Phosphoproteomic studies have revealed that Protein Kinase D1 (PrKD1), another member of the serine/threonine kinase family, is the only kinase known to phosphorylate CDK12 at serine 681 (s681) and serine 685 (s685). While there is an increasing body of literature on CDK12 downstream signaling, there is almost no published data on upstream effectors or regulation of CDK12. Using site-directed mutagenesis, we generated CDK12 s681a and s681a+s685 mutants and transfected them into stable C4-2 and C4-2-PrKD1 cell lines to evaluate the interaction between CDK12 and PrKD1. Immunoprecipitation and immunoblotting revealed that endogenous CDK12 is stable in C4-2 cells with known levels of PrKD1 expression. However, stable transfection of C4-2-PrKD1 cells with wild-type CDK12 resulted in the degradation of nascent CDK12, whereas the non-phosphorylated mutants (s681 and s681+s685) remained stable. Our results suggest that PrKD1-mediated phosphorylation of s681and/or s685 residues affect CDK12 stability in C4-2-PrKD1 cells. We confirmed this finding by treating C4-2-PrKD1 cells with MG-132, a 26 S proteasome inhibitor, and repeating the experiment. Proteasomal inhibition rescued nascent transfected CDK12 levels even after PrKD1 phosphorylation. Our study demonstrates that CDK12 degradation might be mediated by PrKD1 phosphorylation at s681 and/or s685. Because PrKD1 and CKD12 are dysregulated across cancers, the results herein presented have potential implications for the treatment of not only prostate cancer but also other human malignancies.

Citation Format: Victor Chalfant, Carlos Riveros, Sanjeev Shukla, Teruko Osumi, K Balaji. Signaling of cyclin-dependent kinase 12 (CDK12) in prostate cancer cell lines [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 1622.
Abstract 1626: The orthotopic mouse study of cetuximab and dasatinib treatment on BRAF mutated colon cancer cells
Authors: Weng Yu-Cheng , Li Yu-Lin , Hou Xiang-Ling , Koo Hui Min , Lu Yi-Wen , Winarso Viriya Adhiguna , Lin Chia-Wen , Chao Wei-Ting
Published: June 2022

Colorectal cancer is one of the most common cancer in the world. The mortality rate is the third among the top ten cancers. Among colorectal cancer patients, about 65-75% of patients have overexpression of epithelial growth factor receptors (EGFR). The antibody drug cetuximab that inhibits EGFR has also been developed. Moreover, overexpression of src protein is found in about 80% of patients with colorectal cancer. Higher levels of Src expression is related to the metastasis of colon cancer. Dasatinib is an oral taken tyrosine kinase inhibitor to inhibit the src phosphorylation. Therefore, in this study, the mechanism of cetuximab and dasatinib combined treatment in the orthotopic mouse model is investigated. The in vivo mechanism of EGFR and E-cadherin crosstalk in the mouse model is discussed. In this study, HT-29 colon cancer cells with BRAF V600E mutation were orthotopically injected into the cecum wall of BALB/c nude male mice. Two weeks later, cetuximab, dasatinib, and combined treatment of cetuximab and dasatinib were applied for three weeks. The tumor sizes were then recorded after sacrificed. Tumor Tissue were processed for tissue section with H&E staining. Immunohistochemistry (IHC) was applied to detect EGFR, p-Src, E-cadherin with specific antibodies. The protein expression levels were achieved by Western blot. In the results, the tumor size was smaller in cetuximab and dasatinib combined treated group. In IHC staining, the E-cadherin expression were not apparent in primary tumor site, however, were abundant in migrated cells. P-src expression was reduced in the dasatinib treated group and combined treatment group. In western blot result, the E-cadherin was increased after the treatment of dasatinib, and the p-src expression was inhibited after dasatinib treatment and combined treatment. Our results demonstrated cetuximab combined with dasatinib has the better result of suppression of BRAF mutated colon cancer cell growth and metastasis in the orthotopic mouse model.

Citation Format: Yu-Cheng Weng, Yu-Lin Li, Xiang-Ling Hou, Hui Min Koo, Yi-Wen Lu, Viriya Adhiguna Winarso, Chia-Wen Lin, Wei-Ting Chao. The orthotopic mouse study of cetuximab and dasatinib treatment on BRAF mutated colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1626.
Abstract 1628: Application of conditionally reprogrammed cell in cancer model establishment and new target identification
Authors: Long Yuan , Li Le , Zhao Shizhu , Xi Song , Wen Danyi
Published: June 2022

Tumor cell immortalization through conditional reprogramming, particularly from limited biopsy specimen, is an invaluable tool to generate propagating tumor cells for cell-based drug sensitivity assay and bio-banking in vitro. We have successfully reprogrammed primary tumor cells from a number of tumor types, including lung carcinoma, breast cancer and glioma .etc. The conditionally reprogrammed cells exhibited typical colonized growth, which is well maintained upon cryopreservation. In some cases, the cells can be passaged for multiple times and likely become stable cell lines. We attempt to extend conditional reprogrammed tumor cells to reconstitute and grow PDX models in vivo and find the tumor formation rate is much higher than the primary tissue’s. This attempt provides a new process for establishing PDX models from variant types of cancer, especially the cancer types with low success rate in PDX establishment. We also establish PDX-derived cell lines by conditional reprogramming, which could be used in in vitro drug efficacy study and anti-cancer drug target identification. Functional library screening, such as genome-wide CRISPR/Cas9 library or specific pro-siRNA library screening, could discover new targets directly by evaluating the effect of the knockout/knockdown of specific genes. We have established several cell lines from drug resistant PDX, including Osimertinib and other targeted drugs resistant models, and found potential targets by pro-siRNA library screening. These potential targets could be validated on the matching cell line and PDX and the matching in vitro and in vivo tools will also enhance the further research of new targeted small molecules or antibodies.

Citation Format: Yuan Long, Le Li, Shizhu Zhao, Song Xi, Danyi Wen. Application of conditionally reprogrammed cell in cancer model establishment and new target identification [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 1628.
Abstract 163: Regulation of periostin by DDR2 in cancer-associated fibroblasts controls tumor invasion and metastasis in ovarian cancer
Authors: Akinjiyan Favour A. , Dave Ritu M. , Fuh Katherine C.
Published: June 2022

Ovarian cancer has the highest mortality of all gynecological malignancies. As such, there is a need to decipher molecular mechanisms that underlie tumor metastasis in ovarian cancer. Increased expression of receptor tyrosine kinase, DDR2, has been associated with worse patient survival. Identifying downstream targets of DDR2may allow specific modulation of ovarian cancer metastatic pathways with few off-target effects. Additionally, stromal cells play a critical role in metastasis. The crosstalk between tumor and stromal cells cans hift the balance between tumor progression or inhibition. We identified periostin to be highly expressed by fibroblasts expressing DDR2compared to fibroblasts without DDR2(Fold change=2.0, P=0.03).We show that DDR2regulates periostin in ovarian cancer associated fibroblast (CAFs). DDR2and periostin expression in the CAFs leads to512% increase in mesothelial cell clearance ratio(P=0.0004) and matrigel invasion(612vs 193invading cells/hpf, P<0.0001). Mice injected withCAFs expressing periostin as well as ovarian cancer tumor cells have 72% increased tumor burden compared to mice with CAFs that do not express periostin (P=0.006). High periostin expression in CAFs leads to decreased T-cell proliferation and cytotoxicity. We validate periostin as a marker for patient outcome in ovarian cancer patients. Patients with high periostin expression have worse overall survival thant hose with low periostin expression. Mapping regulatory networks involved in tumor initiation and progression is important for identification of novel therapeutic targets.

Citation Format: Favour A. Akinjiyan, Ritu M. Dave, Katherine C. Fuh. Regulation of periostin by DDR2 in cancer-associated fibroblasts controls tumor invasion and metastasis in ovarian 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 163.
Abstract 1641: Evaluating in vivo efficacy of bi-functional fusion protein anti-PD-1-IL21 in humanized B-hIL21R mice and B-hPD-1/hIL21R mice
Authors: Lyu Ruili , Li Zhixia , Kokabee Leila , Yao Jiawei , Nie Yanhui , Guo Chaoshe
Published: June 2022

Interleukin 21 (IL21) binding to its receptor IL21R, results in downstream activation of multiple signaling molecules, including JAK1, JAK3, STAT1, and STAT3. Intriguingly, IL21 has been implicated in both positively and negatively regulating immune responses in a cell-type specific manner, suggesting drug candidates that target IL21/IL21R signaling has therapeutic potential. Specifically, recent reports suggest that antibodies targeting PD-1 fused to IL-21 has a synergistic anti-tumor effect. To generate a suitable animal model for in vivo assessment of a human anti-PD-1-IL21 fusion protein, Biocytogen developed humanized PD-1 and IL21R mice (B-hIL21R mice and B-hPD-1/hIL21R mice) by replacing the murine Il21r and PD-1 gene with the human IL21R and PD-1 counterparts via homologous recombination. Our data shows that human IL21R was detectable in sorted cells from homozygous B-hIL21R and B-hPD-1/hIL21R mice, while human PD-1 was detectable in B-hPD-1/hIL21R mice compared to wild type mice. Furthermore, STAT3 phosphorylation in splenocytes was successfully induced with mouse IL21 or human IL21 in homozygous B-hIL21R mice. In addition, percentages of T cells, B cells, NK cells, DCs, granulocytes, monocytes, macrophages, CD8+ T cells, CD4+ T cells and Tregs in homozygous B-hIL21R mice and B-hPD-1/hIL21R mice were similar to those in wild type mice, demonstrating that introduction of human IL21R and PD-1 genes in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types. To evaluate in vivo efficacy of anti-mPD-1-hIL21 and anti-hPD-1-hIL21 fusion proteins, mouse colon adenocarcinoma MC38 cells were subcutaneously implanted into homozygous B-hIL21R mice (female, 6-7-week-old, n=6) or B-hPD-1/hIL21R mice (female, 6-7-week-old, n=6). Mice were grouped and treated when tumor volume reached approximately 100 mm3. Results showed that anti-PD-1-hIL21 fusion proteins were efficient at inhibiting tumor growth in B-hIL21R and B-hPD-1/hIL21R mice. Altogether, B-hIL21R and B-hPD-1/hIL21R mice are promising humanized mouse models for preclinical in vivo studies to assess novel drug candidates. Keywords: IL21/IL21R, humanized mice, fusion protein

Citation Format: Ruili Lyu, Zhixia Li, Leila Kokabee, Jiawei Yao, Yanhui Nie, Chaoshe Guo. Evaluating in vivo efficacy of bi-functional fusion protein anti-PD-1-IL21 in humanized B-hIL21R mice and B-hPD-1/hIL21R mice [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 1641.
Abstract 1672: Effect of BCOR loss in Xenopus laevis and tropicalis: Insights for retinal development and retinoblastoma
Authors: Zhang Michelle Garying , Owens Dawn , Jones Nicole , Pelaez Daniel , Harbour J. William
Published: June 2022

Retinoblastoma is the most common pediatric eye cancer, accounting for approximately 11% of cancers within the first year of life. Almost all retinoblastoma tumors are initiated by bi-allelic mutational inactivation of RB1, yet there is a broad range of clinical presentations, suggesting the existence of secondary driver events in cancer progression. Genomic analysis identified BCOR as the second most commonly mutated gene in retinoblastoma. There is growing evidence that inactivating BCOR mutations are associated with more aggressive features, such as dedifferentiation, vitreous seeding, optic nerve invasion, and worse patient outcomes. Unfortunately, little is known about the function of BCOR in the retina and the effects of BCOR loss in retinoblastoma. Here, we used Xenopus laevis and tropicalis models to study the role of BCOR in normal ocular and retinal development. In both models, targeted depletion of BCOR in cell lineages giving rise to the eye resulted in a discrete microphthalmia phenotype. Furthermore, we developed in situ hybridization tools for probing characteristic retinal lineage-specific genes and performed histology to compare structural differences. We provide evidence that BCOR normally regulates critical steps in retinal developmental, suggesting that loss of BCOR in retinoblastoma may drive tumor progression by disrupting this function.

Citation Format: Michelle Garying Zhang, Dawn Owens, Nicole Jones, Daniel Pelaez, J. William Harbour. Effect of BCOR loss in Xenopus laevis and tropicalis: Insights for retinal development and retinoblastoma [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 1672.
Abstract 1677: Transcriptomic landscape of medulloblastoma reveals pathways of tumor-stroma remodelling
Authors: Tsea Ioanna , Ruchiy Yana , Verhoeven Manouk , Sinha Indranil , Blomgren Klas , Carlson Lena Maria , Johnsen John Inge , Dyberg Cecilia , Baryawno Ninib
Published: June 2022

Medulloblastoma is one of the most common pediatric malignant brain tumors, currently comprising four distinct molecular subgroups: wingless [WNT], sonic hedgehog [SHH], and groups 3-4. Efforts to identify the mechanisms of medulloblastoma development have focused on mapping the extent of the tumor cell heterogeneity within each subgroup. Nevertheless, little is known about the role of the tumor microenvironment (TME) in medulloblastoma progression, particularly in subgroups 3-4, which have the worst prognosis due to metastatic disease. In this study, we performed single-cell transcriptomics on 14 human medulloblastoma samples spanning all molecular subgroups, to uncover novel TME-tumor interactions modulating medulloblastoma progression and metastasis. Unsupervised clustering of all medulloblastoma samples revealed 18 subclusters, including tumor granule neuron progenitors (GNPs) in different stages of differentiation, stromal cells (including oligodendrocyte precursors, mature oligodendrocytes, astrocytes, fibroblasts, endothelial cells and pericytes), myeloid cells (including microglia, monocytes and macrophages) as well as lymphoid cells. To investigate subgroup-specific signatures as well as TME-tumor pathways, we analyzed each subgroup separately. Analysis of supporting stroma cells in groups 3-4 demonstrated the presence of 8 different stroma populations, including a population of tumor-associated endothelial cells. Tumor-associated endothelial and fibroblast populations of groups 3-4 showed the highest expression of genes for vascular remodeling and extracellular matrix degradation, suggesting an active reprogramming of the stroma by tumor GNP cells to support medulloblastoma progression. Epithelial-to-mesenchymal transition-like processes that regulate stem cell, invasion and metastatic properties were upregulated in the tumor GNP populations of groups 3-4 compared to SHH and WNT subgroups. Finally, we highlight the presence of the CXCL1-CXCL5/CXCR2 metastasis-associated axis in groups 3-4 medulloblastomas as a potential therapeutic target. Our findings provide biological insights into TME processes for the different subgroups of medulloblastoma and possible new potential therapeutic avenues.

Citation Format: Ioanna Tsea, Yana Ruchiy, Manouk Verhoeven, Indranil Sinha, Klas Blomgren, Lena Maria Carlson, John Inge Johnsen, Cecilia Dyberg, Ninib Baryawno. Transcriptomic landscape of medulloblastoma reveals pathways of tumor-stroma remodelling [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 1677.
Abstract 1679: Single-cell profiling reveals a conserved myogenic hierarchy in pediatric rhabdomyosarcomas amenable to differentiation therapy
Authors: Danielli Sara G. , Porpiglia Ermelinda , De Micheli Andrea J. , Bechtold Ingrid , Marques Joana G. , Kasper Stephanie , Blau Helen M. , Wachtel Marco , Schäfer Beat W.
Published: June 2022

Rhabdomyosarcoma (RMS) is a highly aggressive pediatric soft tissue cancer, associated with the skeletal muscle lineage. Despite undoubted expression of key myogenic regulatory factors, RMS cells are blocked in a proliferative state and do not complete terminal differentiation. The extent to which the skeletal muscle lineage is represented in RMS tumors, as well as the mechanisms leading to developmental arrest, remain elusive. We therefore aimed to identify RMS subpopulations, understand why RMS cells are stalled in their differentiation path, and determine mechanisms to pharmacologically restore myogenic differentiation. Using single-cell RNA sequencing and mass cytometry analysis, we profiled cells from 14 PDX-derived primary cultures and three cell lines of alveolar (aRMS) and embryonal RMS (eRMS) subtypes. We discovered that both RMS subtypes contain different cell subpopulations distributed along the myogenic lineage. aRMS tumors recapitulate a yet unrecognized branched myogenic trajectory, where progenitor cells either commit to differentiation or give rise to actively cycling myoblasts. Following in vitro exposure to chemotherapy, aRMS cells show compositional shifts towards undifferentiated states, consistent with a model where treatment rewires cellular trajectories. To quantify aRMS cellular states, we developed an automated image-based single-cell approach and applied it to identify pro-differentiating agents among a library of >240 FDA-approved drugs. We identified the RAS pathway as an important mediator of myogenic differentiation in several aRMS cultures, demonstrating the potential for differentiation therapy. Current studies are ongoing to validate these results in vivo and to uncover drug combinations that completely overcome the differentiation block. Taken together, this study reveals possible cellular origins for RMS and identifies a potential novel treatment strategy for aRMS that targets cellular differentiation.

Citation Format: Sara G. Danielli, Ermelinda Porpiglia, Andrea J. De Micheli, Ingrid Bechtold, Joana G. Marques, Stephanie Kasper, Helen M. Blau, Marco Wachtel, Beat W. Schäfer. Single-cell profiling reveals a conserved myogenic hierarchy in pediatric rhabdomyosarcomas amenable to differentiation therapy [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 1679.
Abstract 1687: Cyto R1 Platform enriches samples’ viability for single-cell sequencing and downstream assays
Authors: Hyler Alexandra R. , Cole Alicia M. , Barua Ridi , Dzutsev Amiran K. , Schmelz Eva M.
Published: June 2022

Single-cell level investigations such as DNA and RNA sequencing, protein analyses, and phenotypic studies require an input sample with a high rate of viability (>90%). If too many dead cells are present in the input, degraded proteins and ambient DNA or RNA can increase background noise that may lead to missing identification of crucial targets. Since the cost for single-cell sequencing experiments still remains high, it is critical to ensure the input sample is optimized for high viability to ensure cost-effective and reproducible data. Current bead assays or other sample viability enrichment techniques used in preparation for single-cell analyses typically result in a significant sample loss, sometimes sacrificing 80% of the starting sample, and these preparation steps require multiple passes (> 45 minutes time) and costly kits. These beads or kits can be biased and change the cell population, subject cells to unwanted stresses, and diminish sample integrity with the time needed for preparations. Here, we investigate sample cell viability enrichment on the Cyto R1 Platform, a label-free, cell enrichment, sorting, and recovery platform. At the core, the Cyto R1 uses Cyto Chips, microfluidic technologies with electrical fields, to phenotypically enrich and sort various cells based upon cells’ unique physical structures and subcellular features. Thus, the Cyto R1 ensures native cell recovery without any unwanted cell tagging to maximize sample integrity. In this work, a variety of single cell solutions derived from T cell lymphoma tumors, ascites fluid, and organ tissues were enriched for viable cells to obtain a final sample viability over 90%. Additionally, we aimed to achieve a minimum of 50,000 viable cells, a common target for single-cell sequencing, in as rapid a timeframe as possible. In one sample origin tested, murine T cell lymphomas, the average starting viability was 65 ± 6.5%, and the final viability after enrichment on the Cyto R1 Platform was 95.8 ± 1.8%. Similarly, murine ascites cell mixtures were enriched from average viabilities below 55% up to 91.6 ± 4.2%. For all samples tested, >50,000 viable cells were obtained in under 30 minutes. This work demonstrates that the Cyto R1 can effectively and efficiently enrich a variety of starting samples for high viability (>90%) as required for downstream assays and single-cell sequencing.

Citation Format: Alexandra R. Hyler, Alicia M. Cole, Ridi Barua, Amiran K. Dzutsev, Eva M. Schmelz. Cyto R1 Platform enriches samples’ viability for single-cell sequencing and downstream assays [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 1687.
Abstract 1710: Spatial transcriptomics and multiplexed imaging to explore tumor heterogeneity and immune complexity
Authors: Chen Siqi , Shinkle Andrew , Zhao Yanyan , Mo Chia-Kuei , Houston Andrew , Lal Preet , Herndon John M. , Fields Ryan C. , Gillanders William , Chen Feng , Ding Li
Published: June 2022

Spatial transcriptomics (ST) and CO-Detection by indEXing (CODEX) are becoming the leading transformative approaches for studying spatially resolved tumor heterogeneity and immune complexity. To explore the application of these two cutting-edge technologies and to further investigate the complexities of tumor microenvironment, we applied 10x Visium Gene Expression platform and Akoya CODEX platform on surgical resections from pancreatic ductal adenocarcinoma (PDAC), breast cancer (BRCA), and clear cell renal carcinoma (ccRCC) patients. A total of 30+ protein markers were screened, verified, and conjugated as CODEX antibodies for the 3 types of cancers. Tumor microenvironment was vividly revealed by a set of cancer markers, immune markers, and stroma cells markers on a single ST slide and a single CODEX image. Notably, the distinct immune microenvironment was observed between tumor nests in the neighborhood in terms of cell types, number of cells, and cell distribution patterns. This work highlights the importance of direct visualization for tumor heterogeneity. It also provides a significant insight for understanding tumor complexity and a promising opportunity to benefit personalized medicine.

Citation Format: Siqi Chen, Andrew Shinkle, Yanyan Zhao, Chia-Kuei Mo, Andrew Houston, Preet Lal, John M. Herndon, Ryan C. Fields, William Gillanders, Feng Chen, Li Ding. Spatial transcriptomics and multiplexed imaging to explore tumor heterogeneity and immune complexity [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 1710.