bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2873: Comparison of metastatic and nonmetastatic breast cancer cell survival in glutamine depleted extracellular matrix detached conditions
Authors: Garg Kanika K. , Sheeley Madeline , Teegarden Dorothy
Published: July 2021

For women, breast cancer is the most diagnosed cancer, with metastasis being the primary cause of most breast cancer-related deaths. During metastasis, cells detach from the extracellular matrix (ECM) to move to a secondary site. Unlike normal cells, metastatic cells are resistant to cell death upon ECM detachment. The amino acid glutamine is used as an energy source during metastatic progression via replenishing the TCA cycle. Breast cancer cells have decreased viability in the absence of glutamine, but the effect of glutamine in ECM detached conditions in metastatic cells is not known. We hypothesize that metastatic and non-metastatic cells have a decreased viability in ECM detachment in glutamine deprived conditions. In this study, metastatic MCF10CA1a cells and non-metastatic Harvey-ras oncogene transfected MCF10A-ras cells were employed. As expected, in ECM detached conditions, the MCF10A-ras cells had a greater decrease in viability (58.4%) in comparison to the MCF10CA1a cells (32.7%). However, in glutamine deprived conditions, the MCF10CA1a cells decrease in viability was 15.2% greater compared to the MCF10A-ras cells. mRNA expression of the major glutamine transporter, SLC1A5, was decreased in detached conditions in both cell lines, but to a greater extent in the MCF10A-ras cells (70.04%) compared to the MCF10CA1a cells (20.15%). Additionally, mRNA expression in detached conditions of glutamate dehydrogenase, the enzyme that metabolizes the downstream glutamine metabolite glutamate to α-ketoglutarate (αKG), was decreased by 77% in the non-metastatic cells, but increased by 23% in the metastatic cells. Additionally, mRNA expression of GOT1, a gene which encodes for the transaminase enzyme which synthesizes αKG and aspartate from glutamate and oxaloacetate, decreased 73% in detached conditions in the non-metastatic cells but was unchanged in the metastatic cells. Moreover, following detachment the glutamine synthesizing enzyme, glutamine synthetase mRNA expression was increased by 81.92% in the non-metastatic cells and 88.43% in the metastatic cells. In summary, the greater effect of glutamine depletion on viability, and the maintained expression of glutamine metabolizing enzymes in the metastatic cells suggest greater dependence on glutamine metabolism in ECM detached conditions compared to non-metastatic cells. Comparison of metastatic and non-metastatic breast cancer cells will provide insights to direct the development of new targets for the prevention of breast cancer metastasis.

Citation Format: Kanika K. Garg, Madeline Sheeley, Dorothy Teegarden. Comparison of metastatic and nonmetastatic breast cancer cell survival in glutamine depleted extracellular matrix detached conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2873.
Abstract 2882: Coronin 1B overexpression promotes invasion of HPV- HNSCC
Authors: Allen Jessica L. , Walk Elyse L. , Rhodes Kristen L. , Markwell Steven M. , Papenberg Brenen W. , Wu Hong , Garcia Marileila , Bear James E. , Weed Scott A.
Published: July 2021

Introduction: Amplification of chromosome 11q13 is the most common DNA aberration in human papilloma virus negative (HPV-) head and neck squamous cell carcinoma. (HSNCC). The 11q13 region encodes various genes known to enhance invasion and drive tumor progression. CORO1B (coronin 1B) flanks the core 11q13 region and is amplified in 7-14% of HNSCC cases. Coronin 1B governs cell motility in fibroblasts by negatively regulating the ability of the Arp 2/3 nucleator cortactin to stabilize the actin cytoskeleton during mesenchymal cell migration. This study seeks to determine the impact of CORO1B amplification and protein overexpression in driving HNSCC progression.

Experimental Procedures: Two patient cohorts were used to assess the impact of CORO1B amplification or overexpression on patient outcome. In addition, HNSCC cell lines lacking or overexpressing coronin 1B were assayed for invadopodia function using gelatin degradation assays. Three-dimensional invasion was analyzed using tumor spheroid and organotypic assays. Invasive and cervical lymph node metastatic potential of CORO1B was evaluated using knockdown and overexpression cells in orthotopic tongue models.

Data Summary: CORO1B amplification and overexpression in patient tumors correlates with enhanced patient decline. Coronin 1B-null cell lines display decreased invadopodia function, with a 30-60% decrease in extracellular matrix-mimicking gelatin degradation. HNSCC tumor spheroids lacking coronin 1B expression decreased 3D invasion in native collagen I, while overexpression enhanced invasive capabilities in both tumor spheroids and organotypic cultures. Coronin 1B knockdown cells show decreased lymph node metastasis, while mice with tumors overexpression coronin 1B have enhanced lymph node metastasis and decreased survival. CORO1B amplification protein overexpression predict poor overall patient survival.

Conclusion: The identification of coronin 1B overexpression as an important mediator in promoting HNSCC invasive processes may define a novel aggressive subset of locoregional involved HNSCC, warranting enhanced clinical intervention.

Citation Format: Jessica L. Allen, Elyse L. Walk, Kristen L. Rhodes, Steven M. Markwell, Brenen W. Papenberg, Hong Wu, Marileila Garcia, James E. Bear, Scott A. Weed. Coronin 1B overexpression promotes invasion of HPV- HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2882.
Abstract 2883: Theranostics nanoparticles targeting to metastatic non small cell lung cancer
Authors: LI LI-JIE , Chan Ming-Hsien , Chang Wei-Min , Hsiao Michael
Published: July 2021

Non-small-cell lung cancer (NSCLC) accounts for one of the most deadly cancers due to metastasis after therapy. Cancer metastasis usually results from epithelial-to-mesenchymal transition (EMT), and metabolic disorder is a key mediator of lung cancer EMT. The linkage between metabolic disorder and NSCLC EMT is still unclear. Using in silico analysis, C4ST1, a key enzyme of glycosaminoglycan synthesis pathway was up-regulation in mesenchymal type NSCLC cells. C4ST1 catalyzes chondroitin sulfate A (CsA) formation. We found both C4ST1 and CsA were up-regulation in mesenchymal type NSCLC cells. Not only C4ST1 overexpression but also CsA treatments changed NSCLC EMT balance and decreased the transcriptional activity on CDH1 promoter region. Furthermore, both treatments could promote in vitro NSCLC migration and invasion abilities in the epithelial type NSCLC cells. Up-regulation of C4ST1 cells also promoted lung cancer metastasis. Moreover, we used CsA binding peptide as a targeting ligands of metastatic NSCLC cells on theranostics nanoparticles. In summary, this study finds key gene in NSCLC EMT. Up-regulation of C4ST1 promotes NSCLC EMT and cell surface CsA content which may provide a novel theranostics niche on metastatic NSCLC cells.

Citation Format: LI-JIE LI, Ming-Hsien Chan, Wei-Min Chang, Michael Hsiao. Theranostics nanoparticles targeting to metastatic non small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2883.
Abstract 2892: Silencing cancer-neuronal crosstalk suppresses perineural invasion in colorectal cancer
Authors: Knoblich Colewyn , Rezvani Khosrow
Published: July 2021

Colorectal cancer (CRC) is one of the most diagnosed cancers and is a leading cause of cancer related mortality. Metastasis away from the primary site of CRC creates further complications, resulting in poor prognosis and decreased overall survival of patients. Perineural invasion (PNI) is an emerging field in cancer research that involves interactions between cancer and the nervous system as an alternative route for metastasis. Various studies have been conducted to understand the underlying mechanisms for metastasis, but PNI has been relatively understudied. Accumulating evidence indicate several signaling pathways are key players in the dynamic crosstalk between cancer cells and the nervous system portending the poor prognosis of patients facilitated by PNI. We hypothesize that colon and rectum cancer cells bi-directionally communicate with their adjacent nervous system influencing cancer progression and metastasis. We are using a combination of in vitro and animal models to define mechanisms underlying PNI in CRC. There are two set of experiments in this study as follow: Aim 1 determines whether neuron- cell lines co-cultured with diverse human colon and rectum cancer cells employed both selective soluble and exosome-based communications to have a stimulatory effect on the migration of colon and rectal cancer cells. We use a set of immunocytometry, western blot, flow-cytometry and proteomic approaches to determine key factors selectively used by colon/rectal cancer cells and neuronal cells to promote downstream metastatic pathways. Aim 2 uses a colon or rectal sciatic NIH-III immunodeficient mouse model to study the influence of the nervous system on cancer cells progression and involved signaling pathways in animal model. Completion of this study will uncover the soluble and vesicle factors released by cancer cells and nerve endings during cancer progression and their potential relevance in the treatment of CRC cancer.

Citation Format: Colewyn Knoblich, Khosrow Rezvani. Silencing cancer-neuronal crosstalk suppresses perineural invasion in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2892.
Abstract 2893: Transmembrane Chloride Intracellular Channel 1 (tmCLIC1) protein as a potential target for colorectal cancer metastasis
Authors: Cianci Francesca , Carlini Valentina , Verduci Ivan , Ranucci Matteo , Mazzanti Michele
Published: July 2021

Colorectal cancer (CRC) is the third most common tumor worldwide and the fourth cause of cancer-related death. The presence of distal metastasis at the time of the first diagnosis represents a worst prognosis factor for patients' long-term survival. Therefore, finding new targets to arrest the metastatic process is a main challenge for successful CRC therapy. Our laboratory focused its attention on the Chloride Intracellular Channel 1 (CLIC1), a metamorphic protein able to switch from a cytoplasmic to a transmembrane form associated to ion channel activity. Previous experiments demonstrated that transmembrane CLIC1 (tmCLIC1) functional activity is negligible in normal colon cells, while increases proportionally to CRC cells' aggressiveness. Moreover, we also revealed that tmCLIC1 supports CRC cells abnormal proliferation rate. Here, we show that tmCLIC1 pharmacological inhibition or its stable knock-down by gene silencing led to a significative reduction of CRC cells migrative and invasive potential, in vitro. To further confirm our hypothesis, we assessed in CLIC1 knock-down cells the downregulation of metalloproteinase-7 (MMP-7) protein and p38 mitogen-activated protein kinase (MAPK) signaling, both involved in migration and metastatic processes. Moreover, by injecting subcutaneously transfected cell lines in nude mice, we demonstrate that CLIC1 knock-down impairs tumor growth and limits cancer cells dissemination to lung. Our results suggest a role for tmCLIC1 in promoting CRC metastasis, making this protein a promising pharmacological target to arrest or slow-down CRC metastasis development. Moreover, thanks to its functional expression exclusively associated with malignant progression, tmCLIC1 could allow to limit side effects on healthy cell population.

Citation Format: Francesca Cianci, Valentina Carlini, Ivan Verduci, Matteo Ranucci, Michele Mazzanti. Transmembrane Chloride Intracellular Channel 1 (tmCLIC1) protein as a potential target for colorectal cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2893.
Abstract 2897: The metastatic potential of high-grade serous ovarian cancer cells along disease progression
Authors: Ritch Sabrina J. , Goyeneche Alicia A. , Noman Abu S. , Telleria Carlos M.
Published: July 2021

Epithelial ovarian cancer (EOC) represents 90% of all ovarian tumors and is considered to be the deadliest gynecological disease. High grade serous ovarian cancer (HGSOC) is the most aggressive form of EOC and represents 70% of all deaths from the disease. Due to a lack of early symptoms and valid screening methods, HGSOC is often diagnosed at late stages, when metastasis has already occurred. HGSOC disseminates from the primary tumor as single cells or clusters that typically adheres to the peritoneal organs and wall. The purpose of this study was to characterize the migratory, invasive, and adhesive abilities of HGSOC cells representing different stages of disease evolution. Both wound healing (WH) and Boyden chamber (BC) assays were performed to study the migratory capacity of five HGSOC cell lines; PEO1, PEO4 and PEO6, from a first patient, and PEO14 and PEO23, from a second patient. The cell lines were generated along different stages of the disease (e.g. at presentation, recurrence, and before death), thus representing disease evolution. To study the invasive capacity of each cell line, a layer of extracellular matrix was added to the BC assays. Adhesion assays were performed on fibronectin coated plates. It was found that the migratory and invasive capacities of the HGSOC cell lines decreased along disease evolution in both cellular series. PEO1 and PEO14, two chemo-sensitive cell lines, were found to have the highest migratory and invasive capacities, while their chemoresistant counterparts, PEO4, PEO6 and PEO23, showed a decrease in migration and invasion. Moreover, a distinct migration pattern was found to be conserved in both WH and BC assays. Cell lines representing an early stage disease had a tendency to migrate as individual cells, while cells representing a late stage disease were observed migrating in clusters. Adhesion rates were found to be similar between all cells lines. The results suggest that HGSOC disease progression seems to be associated with reduced migratory and invasive capacities, contradicting the idea that advanced stage EOC cancers have higher metastatic potential.

Citation Format: Sabrina J. Ritch, Alicia A. Goyeneche, Abu S. Noman, Carlos M. Telleria. The metastatic potential of high-grade serous ovarian cancer cells along disease progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2897.
Abstract 2902: Beneficial effect of vactosertib combined with nal-IRI5-FU/LV in pancreatic cancer treatment
Authors: Hong Eunji , Park Sujin , Oshima Akira , Heo Jinsun , Park Jinah , Lee Jihee , Pang Kyoungwha , Han Songee , Park Hyeyeon , Kim Seong-Jin
Published: July 2021

Pancreatic cancer is one of leading causes of cancer-related mortality in the world. Treatment of pancreatic cancer is difficult due to aggressiveness, distal metastasis, and genetic and epigenetic alterations. Especially, TGF-β is highly expressed in the pancreatic cancer patients, and it leads aggressive tumor metastasis. Regulation of TGF-β signaling is essential for controlling tumor progression and metastasis because TGF-β is involved in cell proliferation, epithelial-to-mesenchymal transition, angiogenesis, metastasis, and immune responses in cancer. Therefore, inhibition of TGF-β signaling may be the key concept of effective chemotherapies for metastatic pancreatic cancer. Recently, nanoliposomal iriotecan (nal-IRI), 5-flourouracil (5-FU) and leucovorin (LV) adjuvant therapy has been applied for pancreatic cancer patients' treatment. Vactosertib, one of the leading TGF-β inhibitor, has been used for clinical trials in many chemotherapy strategies. In this study, we investigated the combination treatment effect of nal-IRI/5-FU/LV and vactosertib on pancreatic cancer in syngeneic orthotopic pancreatic mouse model. The combination treatment significantly increased survival rate compare with control or single treatment of vactosertib or nal-IRI/5-FU/LV. In the histological analysis showed that cancer cell invasion to surrounding tissues was markedly reduced in vactosertib and combination treatment groups. In the RNA-sequencing analysis of tumor RNA, the apoptotic process are positively regulated in vactosertib plus nal-IRI/5-FU/LV group. Furthermore, we found the up-regulated differentially expressed gene (DEG), CCDC80, involved in cell migration, invasion and apoptosis. We ectopically overexpressed CCDC80 on pancreatic cancer cell lines and demonstrated the tumor suppressive role of CCDC80. In conclusion, the combination treatment with vactosertib with nal-IRI/5-FU/LV improved pancreatic cancer survival by inhibiting tumor invasion and inducing apoptosis. These results indicate that combination therapy can be applied to clinical trials in pancreatic cancer patient. This study supported by KHIDI grant (HI14C2640) and National R&D Program for Cancer Control, Ministry for Health and Welfare, Republic of Korea (HA17C0037).

Citation Format: Eunji Hong, Sujin Park, Akira Oshima, Jinsun Heo, Jinah Park, Jihee Lee, Kyoungwha Pang, Songee Han, Hyeyeon Park, Seong-Jin Kim. Beneficial effect of vactosertib combined with nal-IRI5-FU/LV in pancreatic cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2902.
Abstract 2909: Tumor microbiota profiles are associated with molecular subtype and survival in colorectal cancer patients
Authors: Li Fangyu , White Michael G. , Davis Jennifer , Hoffman Kristi L. , Menter David , Ajami Nadim , Zhang Xiaotao , Morris Jeffrey S. , Jenq Robert R. , Petrosino Joseph , Wargo Jennifer A. , Kopetz Scott , Daniel Carrie R.
Published: July 2021

Background: The intestinal microbiome is intimately involved in the pathogenesis of colorectal cancer and likely holds further insights to improve the treatment and management of this deadly disease. In a clinical cohort of patients with colon and rectal cancers, we characterized the tumor microbiota of surgical specimens and evaluated associations with prognostic factors, consensus molecular subtypes (CMS), and survival.

Methods: In 167 patients diagnosed with stage II through IV colon and/or rectal cancer who underwent evaluation and surgical resection (no prior systemic therapy) at The University of Texas MD Anderson Cancer, we characterized the tumor microbiome via 16S rRNA gene sequencing. Each patient's tumor was classified via CMS, a gene expression-based colorectal cancer classification system; and all patients were prospectively followed for disease progression, recurrence, or death. Microbiota diversity and composition were assessed with regard to clinicopathologic and tumor features; and associations with survival were further evaluated in multivariable Cox proportional hazards models.

Results: Left- vs. right-sided colon tumors were characterized by higher microbial diversity, distinct community features, and increased abundance of Bacteroides and Fusobacterium. CMS1 (microsatellite instability immune) vs. CMS2 (canonical) tumors were characterized by higher Bacteroides and Fusobacterium and lower Escherichia (all P<0.05). Fusobacterium-positive and Escherichia-positive tumors were associated with improved 5-year overall survival [presence vs. absence, multivariable-adjusted HR and 95% CI: 0.43 (0.20-0.93) and 0.32 (0.19-0.78), respectively]. Bacteroides was enriched among stage II/III patients who progressed within 2-years (log-rank p<0.001).

Conclusions: Our findings are consistent with those of other groups suggesting the landscape of the tumor microbiome differs by sidedness and molecular subtype, holding important clues and exploitable targets to improve outcomes in colorectal cancer patients.

Citation Format: Fangyu Li, Michael G. White, Jennifer Davis, Kristi L. Hoffman, David Menter, Nadim Ajami, Xiaotao Zhang, Jeffrey S. Morris, Robert R. Jenq, Joseph Petrosino, Jennifer A. Wargo, Scott Kopetz, Carrie R. Daniel. Tumor microbiota profiles are associated with molecular subtype and survival in colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2909.
Abstract 2919: Novel pre-clinical model to identify genetic modifiers of triple negative breast cancer
Authors: Sipe Laura M. , Korba Emily B. , Lu Lu , Williams Robert W. , Ashbrook David G. , Makowski Liza
Published: July 2021

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype with poor outcomes. This is a grave clinical challenge for the ~30,000 patients diagnosed with this disease every year. Discovering genetic modifiers of differential TNBC vulnerability and disease progression is critical to improving predictive and personalized treatments. We hypothesized that using a well-established recombinant inbred strain, novel genetic modifiers of TNBC risk and aggression will be identified. The C3(1)-T antigen (C3Tag) genetically engineered mouse model (GEMM) recapitulates many facets of human basal-like TNBC to demonstrate promoting effects of exposures on tumor phenotypes. However, GEMM are highly constrained by their inbred genotype and do not allow a robust interrogation of the manner in which individual genetic variation might impact tumor initiation, progression, and response to therapy. Therefore, we developed a novel murine model of TNBC in the background of the largest and best characterized genetic reference population. Systems genetics is used to identify gene candidates. Cross-species comparison of our findings with publicly available human GWAS and genomic databases is an effective approach to validate conserved biologically relevant and targetable pathways. To our knowledge, this is the first study to explore modifier genes for TNBC phenotypes using a systems genetics approach in a GEMM for TNBC. Our results will contribute to significant advances in understanding risk and improving outcomes for breast cancer.

Citation Format: Laura M. Sipe, Emily B. Korba, Lu Lu, Robert W. Williams, David G. Ashbrook, Liza Makowski. Novel pre-clinical model to identify genetic modifiers of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2919.
Abstract 2930: Characterization based on therapeutic target biomarkers of patients-driven xenografts model of gastric-type cervical adenocarcinoma
Authors: Kojima Yuki , Yoshida Hiroshi , Okuya Toshihiro , Okuma Hitomi S. , Nishikawa Tadaaki , Tanioka Maki , Sudo Kazuki , Noguchi Emi , Shimoi Tatsunori , Tanase Yasuhito , Uno Masaya , Ishikawa Mitsuya , Fujiwara Yasuhiro , Yonemori Kan , Kato Tomoyasu
Published: July 2021

The majority of cervical adenocarcinomas are associated with human papillomavirus (HPV) mainly HPV types 18 and 16. Patients with gastric-type endocervical adenocarcinoma (GAS) have an aggressive clinical behavior, resulting in poor prognosis compared to those with HPV-associated usual type adenocarcinoma. Chemotherapy resistance to taxane and carboplatin-based regimen has also been reported, which poses a difficulty in managing GAS patients with metastatic lesion. We established patient-driven xenografts (PDX) of two GAS patients and evaluated protein biomarkers for drug development using immunohistochemistry staining. The pathological findings of the established PDX were confirmed by HE staining and special staining to be similar to the pathological findings obtained from the patients' surgical specimens. PDX was established 78 and 48 days after transplantation of patient tumor tissue into immunodeficient mice, respectively. HER2, HER3, PMS2, MSH6, PanTrk, and ARID1A were stained for both PDX and patient-tumor samples to evaluate biomarkers for therapeutic targets. All staining results were consistent between patient tumor samples and PDX for both patients. Staining results for HER3 showed that both patients' tumors and the corresponding PDXs were 3+. Staining for HER2 was 1+ in both cases. In all PDX and patient-tumor samaples, PMS2, MSH6 and ARID1A showed no loss of protein expression, and PanTrk showed no protein expression. In addition, nine additional tumor tissue specimens from GAS patients diagnosed at our institution were stained for HER3 and HER2. HER3 was 3+ in 3 cases and 2+ in 6 cases, and HER2 was 3+ in 1 case, 2+ in 3 cases and 1+ in 5 cases. The present study showed overexpression of HER2 and HER3, suggesting a therapeutic potential of targeting HER2 and/or HER3 in GAS patients. We are next planning an in vivo study to develop therapies in GAS patients.

Citation Format: Yuki Kojima, Hiroshi Yoshida, Toshihiro Okuya, Hitomi S. Okuma, Tadaaki Nishikawa, Maki Tanioka, Kazuki Sudo, Emi Noguchi, Tatsunori Shimoi, Yasuhito Tanase, Masaya Uno, Mitsuya Ishikawa, Yasuhiro Fujiwara, Kan Yonemori, Tomoyasu Kato. Characterization based on therapeutic target biomarkers of patients-driven xenografts model of gastric-type cervical adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2930.
Abstract 2938: APOBEC3B and TKI resistance in EGFR mutant lung cancer
Authors: Caswell Deborah , Mayekar Manasi , Law Emily , Hill William , Gronroos Eva , Harris Reuben , Bivona Trever , Swanton Charles
Published: July 2021

Lung cancer is the leading cause of cancer-related deaths worldwide. EGFR mutations, which account for about 25% of lung cancer cases, are more commonly associated with lung cancer in never smokers. Although response rates to EGFR tyrosine kinase inhibitors (TKI) used as a first line therapy are high, acquired resistance inevitably occurs. Elevated tumor mutation burden in EGFR mutant lung cancer is associated with a shorter progression free survival, and worse overall survival with both first and second generation TKIs. Increasing our understanding of drivers of mutagenesis in lung cancer is critical in our efforts to prevent tumor reoccurrence and resistance. APOBEC-driven mutagenesis has been shown to occur in multiple cancer types including lung adenocarcinoma. Using the TCGA and TRACERx datasets we have demonstrated that APOBEC3B is significantly upregulated when compared with other APOBEC family members in EGFR mutant lung adenocarcinoma, and that the APOBEC mutation signature is enriched in a subset of patients with EGFR mutant lung adenocarcinoma. To model APOBEC mutagenesis in lung cancer, we have developed a novel lung cancer mouse model, by crossing the ROSA26LSL-APOBEC3B (A3B) mouse model with several different EGFR mutant mouse models. Using these models, we find that APOBEC3B expression is selected for post-TKI and contributes to resistance to TKI therapy.

Citation Format: Deborah Caswell, Manasi Mayekar, Emily Law, William Hill, Eva Gronroos, Reuben Harris, Trever Bivona, Charles Swanton. APOBEC3B and TKI resistance in EGFR mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2938.
Abstract 2945: Novel engineered B-NDG B2M/KO mice plus and steroid treated B-NDG Mice shows delayed GvHD response when engrafted with hPBMC
Authors: Gao Lan , Sharma Shruti , Lin Eugene , Wang Xiaoen , Shang Chengzhang , Hu Yuting , An W. Frank , Chawla Mehma Kaur , Li Kaiqi , Lin Qingcong , Yu Zhaoxue (Luke) , Liu Yujie
Published: July 2021

Severely immunodeficient mouse strains, such as NOD.Cg-Prkdcscid Il2rg-/-, have been used to study the immune response in humans. These mice lack T, B and natural killer (NK) cells and allow for engraftment of human peripheral blood mononuclear cells (PBMCs). However, hPBMC engraftment may lead to severe acute xenogeneic graft versus host disease (GvHD) which arises due to a response from T cells against human leukocyte antigens (HLA) that are encoded by major histocompatibility complex (MHC) class I and II. GvHD shortens animal life span and results in only a brief window of evaluation of human immune cell functions in such mouse models. Efforts have been undertaken to reduce human PBMC-induced GvHD and to subsequently extend the window of human immunity assessment by eliminating mouse MHC I and MHC II. While these beta-2-microglobulin (B2M) knockout mice which are MHC deficient showed fewer symptoms of GvHD, an unintended result was the shorter half life of the antibodies which lead to its rapid clearance. This occurred because B2M is also associated with neonatal Fc receptor (FcRn) and is important to FcRn-mediated in vivo antibody recycling and retention. This is a pharmacokinetic limitation because it renders antibody efficacy assessment and makes these mice unsuitable for studying immune responses. To overcome this limitation, we engineered B-NDG B2M knockout mice plus (NOD.Cg-Prkdcscid Il2rg-/-/Bcgen) that is deficient in MHC I expression but expresses the B2M gene fused in the FcRn gene. In the present study we demonstrate the phenotypic changes in the immune system of the engineered mice using flow cytometry and validate immune tolerance developed over the several weeks of post engraftment by looking at the homeostatic balance between CD8/Treg ratio in the partially reconstituted humanized immune systems. In a parallel study, we used corticosteroid treatment in hPBMC induced B-NDG mice GvHD model. Corticosteroids are the fine line of therapy for GvHD and the phenotypic response generated in B-NDG mice confirms this finding in our GvHD model.

Citation Format: Lan Gao, Shruti Sharma, Eugene Lin, Xiaoen Wang, Chengzhang Shang, Yuting Hu, W. Frank An, Mehma Kaur Chawla, Kaiqi Li, Qingcong Lin, Zhaoxue (Luke) Yu, Yujie Liu. Novel engineered B-NDG B2M/KO mice plus and steroid treated B-NDG Mice shows delayed GvHD response when engrafted with hPBMC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2945.
Abstract 2951: Anti-GCGR in vivo efficacy evaluated in a novel humanized B-hGCGR mouse model
Authors: Zhang Rufeng , Shang Chengzhang , Hu Yuting , Chromikova Veronika , Lin Qingcong
Published: July 2021

GCGR (glucagon receptor) is a G-protein-coupled seven-transmembrane protein, a typical representative of the class B GPCR family. Animals homozygous for a targeted mutation in this gene exhibit reduced blood glucose levels, increased plasma glucagon and amino acid levels associated with alpha-cell hyperplasia. Glucagon activates intracellular adenylate cyclase by specifically binding to GCGR on the surface of target cells in the liver. This results in an increase in intracellular cAMP levels, playing a role in promoting glycogenolysis and gluconeogenesis, and promoting blood glucose elevation. The crucial role in regulation of blood glucose levels and glucose homeostasis is posing GCGR as a potential drug target for type 2 diabetes and related diseases. Currently, much attention and many resources are directed towards research and development of antibody drugs targeting the glucagon receptor. Joining these efforts, Biocytogen has developed a GCGR humanized mouse model (B-hGCGR mouse). Human GCGR protein and mRNA were detected in liver tissue of homozygous B-hGCGR mice, but not in wild type mice. Physiological indexes have been analyzed. Results indicated that the random blood glucose, fasting blood glucose, and glucose tolerance were comparable to the wild type mice. In addition, we characterized the ability of glucagon to stimulate production of cAMP. We observed that glucagon elicits a dose specific increase of cAMP in membranes prepared from the humanized GCGR mice comparable to the cAMP levels observed in membranes prepared from wild type mice. In pharmacodynamic experiments using B-hGCGR mice we showed that an anti-GCGR antibody (Crotedumab) was able to effectively reduce random blood glucose and fasting blood glucose levels and improve the glucose tolerance, validating our humanized mouse model. This antibody also blocked the functional response to glucagon in B-hGCGR mice more efficiently than what can be observed in an approach targeting the wild type murine receptor using antagonists. These data demonstrate that B-hGCGR mice are a promising model for preclinical in vivo pharmacodynamic assessment of anti-GCGR antibodies.

Citation Format: Rufeng Zhang, Chengzhang Shang, Yuting Hu, Veronika Chromikova, Qingcong Lin. Anti-GCGR in vivo efficacy evaluated in a novel humanized B-hGCGR mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2951.
Abstract 2953: Humanized IL6/hIL6R mouse model supports the evaluation of therapeutic drug candidates targeting to human IL6 and IL6 receptor
Authors: Zhao Lei , Mao Yang , Wang Linlin , Chen Yichao , Kokabee Leila , Lin Qingcong
Published: July 2021

Interleukin 6 (IL-6) gene encodes a cytokine that functions in inflammation and the maturation of B cells and is a potent pleiotropic cytokine that regulates cell growth and differentiation and plays an important role in the immune response Evidence of recent research shows blockade of IL-6 and IL-6 receptor interaction can offer a broader therapeutic strategy for various diseases included in acute systemic and chronic inflammatory diseases. The development of humanized mouse model can greatly support preclinical evaluation of human IL6 and IL6 receptor therapeutic drugs. To evaluate the efficacy of IL-6 and IL-6R therapeutic drug candidates, Biocytogen has generated a double humanized B-hIL6/hIL6R mouse model. In this model, the mouse IL6 gene that encodes the full length was replaced with the corresponding human sequences and the CDS of human IL6R gene was inserted into the IL6RA gene in B-hIL6/hIL6R mice. Human IL6 or IL6R protein expression was detected in homozygous B-hIL6/hIL6R mice by species-specific IL6 ELISA kit, also evaluated by flow cytometry. In efficacy study of anti-human IL6 antibodies in B-hIL6/hIL6R mice with collagen induced arthritis (CIA) , the IL6 antibody treatment reversed the disease progression by decreasing the total clinical score compared to the control group. The efficacy suggests that the B-hIL6/hIL6R mouse model is an effective tool for in vivo efficacy study in the development of IL6 and IL6R antibodies which provide a promising potential in future human clinical trials.

Citation Format: Lei Zhao, Yang Mao, Linlin Wang, Yichao Chen, Leila Kokabee, Qingcong Lin. Humanized IL6/hIL6R mouse model supports the evaluation of therapeutic drug candidates targeting to human IL6 and IL6 receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2953.
Abstract 2958: Drug induced resistant tumor models enable the development of next-generation anticancer therapeutics
Authors: Ni Ting , Zhang Zhixiang , Tang Xuzhen , Shi Wenting , Gu Qingyang , Ji Qunsheng
Published: July 2021

Recent advances in cancer biology and diagnosis are providing more targeted approaches to treat cancers. Therapy targeting the specific oncogenic driver could inhibit tumor progression and helps to a favorable prognosis in clinical practice. Recently, the discovery of driver mutations in various cancers such as EGFR, ER, CDK, c–MET, TRK and BTK provides a personalized targeted treatment. However, most patients who initially benefit a lot from targeted therapies eventually become resistant to treatments. Acquired resistance limits the long–term efficacy. The commonly acquired resistant mechanisms include target gene mutation, alternative pathway activation, histological or phenotypic transformation and so on. Although a variety of mechanisms of acquired resistance have been reported, optimal treatment for acquired resistance is not yet clearly defined.

To mimic the clinical resistant after long–term treatment, we established a panel of in vivo induced drug resistant CDX and PDX models by continuous dosing of targeted drugs to mice, covering a series of first–line targeted drugs Tamoxifen, Olaparib, BMN–673, Ibrutinib, Crizotinib, Erlotinib, Palbociclib, Capmatinib, Entrectinib, Cabozantinib, Sorafenib and Irinotecan. Resistant models were derived from sensitive models harboring specific oncogenic driver. Regrowth tumors were continually treated with targeted drugs and passaged several times until a stable drug resistance phenotype occurred. To explore the resistant mechanisms, we performed WES and RNA–seq analysis in the gene expression levels, and western blotting and IHC staining in the protein expression levels. In summary, these drug induced resistant tumor models provide an opportunity to evaluate the next–generation anticancer therapeutics to overcome therapeutic resistance in tumors.

Citation Format: Ting Ni, Zhixiang Zhang, Xuzhen Tang, Wenting Shi, Qingyang Gu, Qunsheng Ji. Drug induced resistant tumor models enable the development of next-generation anticancer therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2958.
Abstract 2964: Immuno-reactive cancer organoid models to examine microbiome metabolite effects on immune checkpoint blockade efficacy
Authors: Shelkey Ethan , Soto-Pantoja David , Lu Yong , Soker Shay
Published: July 2021

Introduction: As the number of available immunotherapies for solid tumors increase, their prevalence in the clinic continues to rise as well. While the results are promising, and immunotherapies have benefits over traditional chemotherapeutics, a sizable percentage of patients are non-responders to all types of immunotherapy. These differences in sensitivity can be either innate or acquired. Yet, there has been limited 3D in vitro models to assess tumor immune-reactivity. These systems are ideal for isolating specific molecular mechanisms that dictate cell behavior and interactions. Our goal was to create an organoid model containing cancer cells paired with cytotoxic T-cells to model immune checkpoint blockade (ICB) efficacy. This model could then be used to examine novel microbiome-ICB interactions shown by recent research to alter therapeutic response levels in patients.

Methods: We created tumor organoids consisting of tumor and immune cells, embedded in extracellular matrix (ECM)-like hydrogels. Organoids were treated with therapeutic equivalent doses of anti-PD-1 and anti-CTLA-4 or single dose of anti-CD-47 and cell viability assays, flow cytometry, RT-qPCR, and immunohistochemistry staining were performed to determine the effects of ICB. Physiologic concentrations of metabolites derived from likely effector bacterial species were then evaluated for their effects on genetic expression and immunotherapy efficacy.

Results and Discussion: We showed that ICB therapy stimulated internally localizing T-cells, inducing T-cell-mediated tumor cell killing. ICB treated samples resulted significant loss of viability with corroborating readings from the other methods of characterization. RT-qPCR resulted in some elucidation of potential cellular changes due to bacterial metabolites with further quantification ongoing.

Conclusion: We have created an ex-vivo tumor immune-reactive organoid model for studying immunotherapy. We can now continue to change individual microenvironment factors, such as microbiome metabolites, and observe their impacts on immunotherapy efficacy to better understand what conditions are conducive or detrimental to successful ICB treatment.

Citation Format: Ethan Shelkey, David Soto-Pantoja, Yong Lu, Shay Soker. Immuno-reactive cancer organoid models to examine microbiome metabolite effects on immune checkpoint blockade efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2964.
Abstract 3006: Melanoma cell lines derived from patient derived xenografts (PDX) can contain murine chromosomes resultant from cell fusions
Authors: Vega-Crespo Agustin , Baselga-Carretero Ignacio , Perez-Garcilaso Ivan , Jilani Sameeha , Chen Kevin , Khullar Vaneet , Medina Egmidio , Teper Yaroslav , Ribas Antoni , Comin-Anduix Begoña
Published: July 2021

Introduction: Patient Derived Xenografts (PDX) provide a distinct platform to generate in vivo and ex vivo cell lines to investigate novel therapies. However, human-murine cell fusions may skew the models and is a poorly characterized phenomenon.

Methods: Malignant melanoma IRB-consented biopsies [IRBs: 17-000548; 17-001685; 17-001351; 11-003254] were excised from various sites and implanted into gender-matched 4-6 weeks old NOD-scid IL2Rgammanull mice. Tumor-bearing mice were humanely euthanized and adherent cell lines were generated after Miltenyi gentleMACS™ dissociation of the PDXs. Briefly, adherent cells are allowed to proliferate and passaged when 70% confluent for approximately 4-8 weeks. PDX-derived cell products were processed for long term-cryostorage and an aliquot was sent for GenePrint 10 System-Promega®-LaragenTM cell line authentication paired with patient-matched peripheral blood mononuclear cells (PBMC) for specimen congruency. Mouse-genome contaminant resulting cultures were prepared and sent for cell line characterization by G-banded metaphase karyotyping-Cell Line GeneticsTM. Cultures containing murine karyotypes were processed according to manufacturer recommendations by Miltenyi Mouse Cell Depletion KitTM where the flow through (negative fraction) represents the human-cell fraction.

Results: We implanted 86 patient-derived biopsies into mice over 3years, from which 39 [45%] resulted in progressively growing PDXs. From these PDXs we have generated 26 [37%]-patient-specific PDX-derived cell lines. G-banded metaphase karyotyping demonstrated that 6 [24%]- cell lines presented murine chromosome contamination. The immunoprofile of these 6 cellular materials appear to be murine as the totality of the cultures were sequestered in the column by mouse depletion cell cocktail. Separation assay was validated by bonafide human cell lines, utilized as human positive controls, M257 and MX210 both with human karyotypes and matched Cell ID profiling. The murine colon carcinoma cell line MC38, was utilized as a positive murine control, and performed identically as the 6 PDX-derived cellular materials with abnormal mouse genomes.

Conclusion: Detection of murine-human fusion cells in cell lines derived from PDXs may limit the utility of these models.

Citation Format: Agustin Vega-Crespo, Ignacio Baselga-Carretero, Ivan Perez-Garcilaso, Sameeha Jilani, Kevin Chen, Vaneet Khullar, Egmidio Medina, Yaroslav Teper, Antoni Ribas, Begoña Comin-Anduix. Melanoma cell lines derived from patient derived xenografts (PDX) can contain murine chromosomes resultant from cell fusions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3006.
Abstract 3019: An ultra-accurate NGS assay reveals great variation in mouse ratios in xenograft tumors by mouse strain and cancer type
Authors: Qian Wubin , Guo Sheng
Published: July 2021

Background: Tumors are infiltrated by stromal cells including fibroblasts, myofibroblasts, macrophages, lymphocytes, mast cells, endothelial cells, etc. Heterotypic interactions, such as ligand-receptor signaling, between the two support the growth and metastasis of cancer cells. Xenograft models are useful in studying such interactions because mouse stromal cells quickly replace the human counterparts associated with human tumors implanted in immunocompromised mice, and can be readily detected. There is, however, a lack of accurate and large-scale studies of human/mouse ratios in xenograft tumors, although there are several ways of estimating mouse ratios in xenograft tumors, including RT-PCR, RNA-seq and whole-exome sequencing (WES). None of these methods is accurate due to some systematic bias. For example, RT-PCR compares the expression of one or a few genes, e.g. GAPDH, between human and mouse by using species-specific primers that have inequivalent amplification efficiency and large errors. We have developed a NGS assay to quantify human/mouse ratios in human-mouse mixtures more accurately than the methods currently employed.

Methods: Our NGS assay simultaneously amplified 108 human and mouse homologous segments, each with identical flanking sequences in human and mouse, so that a pair of common primers was designed to have equal hybridization efficiency in the two species. A total of 5000 tumors from our patient-derived xenograft (PDX) collection were profiled across 1674 models in 24 cancer types.

Results: Several interesting observations were identified. (a) The average mouse content across the collection of PDX models was 23%, but with great variation, ranging from zero to 93%. (b) PDXs have significantly more mouse content in BALB/c than in NOD/SCID mice (31.9% vs 22.1%, p value<2.2e-16). (c) Different cancers have varied mouse content, for example, pancreatic cancer has much higher mouse content than colorectal cancer (37.3±16.6% vs 17.9±12.2%). (d) Different passages of the same PDX model can have very different mouse ratios and this is likely due to randomness in tumor dissection.

Conclusions: Our NGS assay and findings will help the study of stroma and the tumor microenvironment of tumors in xenografts models.

Citation Format: Wubin Qian, Sheng Guo. An ultra-accurate NGS assay reveals great variation in mouse ratios in xenograft tumors by mouse strain and cancer type [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3019.