Injection Of Tumor Cells Research Articles

Establishing Patient-Derived Xenograft (PDX) Models of Lymphomas.

Patient-derived xenograft (PDX) models of lymphoma typically involve the injection of human tumor cells into an immunocompromised murine host. PDXs have the advantage that the tumor cells grow in a 3D environment within the mouse, meaning the selection pressure of in vitro establishment is avoided and the tumor cells better maintain their genetic heterogeneity. Here, we outline a method for producing and maintaining a PDX model of lymphoma. We describe three different methods to isolate a single cell suspension of the primary patient tumor, followed by either subcutaneous or intraperitoneal injection into an immunocompromised mouse. We then detail how to monitor tumor growth and how to harvest, passage, and store the tumors once they have grown. We highlight and discuss important protocol considerations including technical hints as well as the advantages and disadvantages of the methods described.

Flurbiprofen axetil is involved in basal-like breast cancer metastasis via suppressing the MEK/ERK signaling pathway.

Flurbiprofen axetil is commonly utilized in clinical practice as one of the nonsteroidal anti-inflammatory drugs (NSAIDs) and is included in multimodal analgesia regimens postbreast cancer surgery. Numerous NSAIDs have been studied for their potential to both promote and inhibit cancer. Given the variability in their effects on tumors, further investigation into the specific role of flurbiprofen axetil is warranted. Therefore, the primary objective of this study was to assess the impact of flurbiprofen axetil on basal-like breast cancer (BLBC) metastasis and elucidate the underlying molecular mechanisms involved. The BLBC metastasis mouse model was established by caudal vein injection of tumor cells. The lung metastasis of breast cancer in mice and the effect of flurbiprofen axetil were assessed by in vivo bioluminescence imaging, hematoxylin and eosin staining and immunohistochemistry. In vitro, the results of flurbiprofen axetil on the proliferation, migration, and invasion of MDA-MB-231 human breast cancer cells and BT-549 human breast cancer cells were assessed by colony formation assay and transwell assay. The effects of flurbiprofen axetil on several tumor metastasis-related signaling pathway proteins were examined by western blot, and the reversal extent of the flurbiprofen axetil effect by Ro 67-7476 (ERK phosphorylation agonist) was detected by transwell assay. The results showed that flurbiprofen axetil significantly inhibited BLBC lung metastasis in mice. Flurbiprofen axetil similarly inhibited breast cancer cell migration and invasion in vitro but did not affect their proliferation. Mechanistic investigations have revealed that flurbiprofen axetil exerts a noteworthy inhibitory influence on the MEK/ERK pathway while exhibiting no significant alteration in the expression of other pathway proteins intricately associated with epithelial-mesenchymal transition. In conclusion, the inhibitory effect of flurbiprofen axetil on BLBC metastasis is characterized by its selectivity in targeting the MEK/ERK signaling pathway rather than exerting a broad impact on the global signaling pathway.

Towards reliable hyperspectral imaging biomarkers of CT26 murine tumor model

The non-invasive monitoring of tumor growth can offer invaluable diagnostic insights and enhance our understanding of tumors and their microenvironment. Integrating hyperspectral imaging (HSI) with three-dimensional optical profilometry (3D OP) makes contactless and non-invasive tumor diagnosis possible by utilizing the inherent tissue contrast provided by visible (VIS) and near-infrared (NIR) light. Consequently, valuable information regarding tumors and healthy tissues can be extracted from the acquired hyperspectral images. Until now, very few methods have been used to monitor tumor models in vivo daily and non-invasively. In this research, we conducted a 14-day study monitoring BALB/c mice with subcutaneously grown CT26 murine colon carcinomas in vivo, commencing on the day of tumor cell injection. We extracted physiological properties such as total hemoglobin (THB) and tissue oxygenation (StO2) using the inverse adding-doubling (IAD) algorithm and manually segmented the tissues. We then selected the ten most relevant features describing tumors using the Max-Relevance Min-Redundancy (MRMR) algorithm and utilized 30 classic and advanced machine learning (ML) algorithms to discriminate tumors from healthy tissues. Finally, we tested the robustness of feature selection and model performance by smoothing tissue parameter maps extracted by IAD with a variable kernel and omitting selected training data. We could discriminate CT26 tumor models from surrounding healthy tissues with an area under the curve (AUC) of up to 1 for models based on the gradient boosting method, linear discriminant analysis, and random forests. Our findings help pave the way for precise and robust imaging biomarkers that could aid tumor diagnosis and advance clinical practice.

Pan02 pancreatic tumor models carrying the GFP marker in mice

Animal tumor models are used for preclinical studies of drugs and cancer therapy. The aim of this work was to analyze the growth of murine pancreatic tumor cells Pan02, carrying GFP marker, injected subcutaneously (s. c.), intraperitoneally (i. p.) or orthotopically into the pancreas (ortho) of C57BL/6 mice. Mice were injected with 2 × 105 cells: s. c. in the right flank; i. p. with a syringe into the abdominal cavity, or ortho surgically under the pancreas capsule. The weight of mice was determined in the dynamics of tumor growth, and blood serum was taken to analyze the antibody response to the GFP reference protein. At the 2nd and 4th weeks of tumor growth, some mice were slaughtered and the expression of GFP by the tumor cells, as well as the composition of the immune cells in the tumor, were analyzed by flow cytometry and confocal microscopy. It was shown that with the different localization, the pancreatic tumors grew at different rates and lethality. When the tumor was injected i. p., mice lost weight with rapid tumor growth. In the ortho model, the mice increased their weight. Mortality in the s. c. and i. p. groups was comparable. In the s. c. model, the tumor grew slowly to a volume of 200-400 mm3 and stopped growing. There was no mortality in this group during the follow-up period (2 months). The same antibody response to GFP was formed with all injection schemes. The subpopulation composition of immune cells varied greatly in the different models of tumor cell administration. Regardless of the type of immune response, Pan02-GFP cells rapidly suppressed GFP gene expression in vivo. The data obtained showed that murine pancreatic tumor Pan02 is immunogenic and causes the formation of an adaptive immune response. Regardless of the presence or absence of an immune response and elimination of GFP+ cells, the tumor continued to grow in the i. p. and ortho models, but not in the s. c. one, and caused the death of mice. When conducting preclinical studies, it is necessary to use several ways of tumor cell injection to obtain a more objective result.

Platelet Angiopoietin-1 Protects Against Murine Models of Tumor Metastasis.

In addition to their fundamental roles in preserving vascular integrity, platelets also contribute to tumor angiogenesis and metastasis. However, despite being a reservoir for angiogenic and metastatic cytokines, platelets also harbor negative regulators of tumor progression. Angpt1 (angiopoietin-1) is a cytokine essential for developmental angiogenesis that also protects against tumor cell metastasis through an undefined mechanism. Although activated platelets release Angpt1 from α-granules into circulation, the contributions of platelet Angpt1 to tumor growth, angiogenesis, and metastasis have not been investigated. Using cytokine arrays and ELISAs, we first compared platelet Angpt1 levels in breast and melanoma mouse tumor models to tumor-free controls. We then assessed tumor growth and metastasis in mice lacking megakaryocyte and platelet Angpt1 (Angpt1Plt KO). The spontaneous metastasis of mammary-injected tumor cells to the lungs was quantified using RT-PCR (reverse transcription-polymerase chain reaction). The lung colonization of intravenously injected tumor cells and tumor cell extravasation were determined using fluorescent microscopy and flow cytometry. Platelet Angpt1 is selectively upregulated in the PyMT (polyoma middle tumor antigen) breast cancer mouse model, and platelets are the principal source of Angpt1 in blood circulation. While primary tumor growth and angiogenesis were unaffected, Angpt1Plt KO mice had both increased spontaneous lung metastasis and tumor cell lung colonization following mammary or intravenous injection, respectively. Although platelet Angpt1 did not affect initial tumor cell entrapment in the lungs, Angpt1Plt KO mice had increased tumor cell retention and extravasation. Serum from Angpt1Plt KO mice increased endothelial permeability and reduced VE (vascular endothelial)-cadherin expression at endothelial junctions compared with serum from control mice (Angpt1WT). Platelets provide an intravascular source of Angpt1 that restrains tumor metastasis by preserving the lung microvasculature to limit tumor cell extravasation.

Downregulation of malic enzyme 3 facilitates progression of gastric carcinoma via regulating intracellular oxidative stress and hypoxia-inducible factor-1α stabilization

BackgroundGastric cancer (GC) is one of the most malignant cancers worldwide. Metabolism disorder is a critical characteristic of malignant tumors related to tumor progression and metastasis. However, the expression and molecular mechanism of malic enzyme 3 (ME3) in GC are rarely reported. In this study, we aim to investigate the molecular mechanism of ME3 in the development of GC and to explore its potential value as a prognostic and therapeutic target in GC.MethodME3 mRNA and protein expression were evaluated in patients with GC using RT-qPCR, WB, and immunohistochemistry, as well as their correlation with clinicopathological indicators. The effect of ME3 on proliferation and metastasis was evaluated using Cell Counting Kit-8 (CCK-8), 5-ethynyl-20-deoxyuridine (EdU) assay, transwell assay, wound healing assay, and subcutaneous injection or tail vein injection of tumor cells in mice model. The effects of ME3 knockdown on the level of metabolites and hypoxia-inducible factor-1α (HIF-1α) protein were determined in GC cells. Oxidative phosphorylation was measured to evaluate adenosine triphosphate (ATP) production.ResultsME3 was downregulated in human GC tissues (P < 0.001). The decreased ME3 mRNA expression was associated with younger age (P = 0.02), pathological staging (P = 0.049), and lymph node metastasis (P = 0.001), while low ME3 expression was associated with tumor size (P = 0.048), tumor invasion depth (P < 0.001), lymph node metastasis (P = 0.018), TNM staging (P < 0.001), and poor prognosis (OS, P = 0.0206; PFS P = 0.0453). ME3 knockdown promoted GC cell malignancy phenotypes. Moreover, α-ketoglutarate (α-KG) and NADPH/NADP+ ratios were reduced while malate was increased in the ME3 knockdown group under normoxia. When cells were incubated under hypoxia, the NADPH/NADP+ ratio and α-KG decreased while intracellular reactive oxygen species (ROS) increased significantly. The ME3 knockdown group exhibited an increase in ATP production and while ME3 overexpression group exhibited oppositely. We discovered that ME3 and HIF-1α expression were negatively correlated in GC cells and tissues, and proposed the hypothesis: downregulation of ME3 promotes GC progression via regulating intracellular oxidative stress and HIF-1α.ConclusionWe provide evidence that ME3 downregulation is associated with poor prognosis in GC patients and propose a hypothesis for the ME3 regulatory mechanism in GC progression. The present study is of great scientific significance and clinical value for exploring the prognostic and therapeutic targets of GC, evaluating and improving the clinical efficacy of patients, reducing recurrence and metastasis, and improving the prognosis and quality of life of patients.

Lead Decreases Bone Morphogenetic Protein-7 (BMP-7) Expression and Increases Renal Cell Carcinoma Growth in a Sex-Divergent Manner.

Both tissue and blood lead levels are elevated in renal cell carcinoma (RCC) patients. These studies assessed the impact of the subchronic lead challenge on the progression of RCC in vitro and in vivo. Lead challenge of Renca cells with 0.5 μM lead acetate for 10 consecutive passages decreased E-cadherin expression and cell aggregation. Proliferation, colony formation, and wound healing were increased. When lead-challenged cells were injected into mice, tumor size at day 21 was increased; interestingly, this increase was seen in male but not female mice. When mice were challenged with 32 ppm lead in drinking water for 20 weeks prior to tumor cell injection, there was an increase in tumor size in male, but not female, mice at day 21. To investigate the mechanism underlying the sex differences, the expression of sex hormone receptors in Renca cells was examined. Control Renca cells expressed estrogen receptor (ER) alpha but not ER beta or androgen receptor (AR), as assessed by qPCR, and the expression of ERα was increased in tumors in both sexes. In tumor samples harvested from lead-challenged cells, both ERα and AR were detected by qPCR, yet there was a significant decrease in AR seen in lead-challenged tumor cells from male mice only. This was paralleled by a plate-based array demonstrating the same sex difference in BMP-7 gene expression, which was also significantly decreased in tumors harvested from male but not female mice; this finding was validated by immunohistochemistry. A similar expression pattern was seen in tumors harvested from the mice challenged with lead in the drinking water. These data suggest that lead promotes RCC progression in a sex-dependent via a mechanism that may involve sex-divergent changes in BMP-7 expression.

Abstract PO2-28-01: Deleting caveolin-1 in epithelial cells increases doxorubicin sensitivity in advance stage of breast cancer

Abstract Caveolin-1 (Cav-1) is a lipid raft protein with a dual role, serving as a tumor suppressor and a promoter. Studies have unveiled Cav-1’s significance in breast cancer progression, impacting various aspects, including cell proliferation, apoptosis, autophagy, invasion, migration, and metastasis. Previously, our research demonstrated that the knockout (KO) of Cav-1 from epithelial cells reduced lung metastasis originating from primary tumors. In this study, we sought to investigate whether Cav-1 KO in epithelial cells increases sensitivity to doxorubicin (Dox) using both in vitro and in vivo studies. In our in vitro study, we observed reduced cell numbers in doxorubicin-treated Cav-1 KO cells compared to Dox-treated 4T1 cells (wild type). Flow cytometry-based cell cycle assay showed a significant increase in G2M phase arrest in Dox-treated Cav-1 KO cells compared with wild-type, confirming reduced cell growth and proliferation. To validate the increased sensitivity to Dox in vivo, we used a syngeneic tumor model using Cav-1 KO and 4T1 wild-type cells injected into the mammary fat of female Balb/c mice intraperitoneally. Subsequently, after one week of tumor cell injection, both groups received Dox at a dose of 8 mg/kg body weight/week for 3 weeks (a cumulative dose of 24 mg/kg body weight). Primary tumor sizes exhibited a significant reduction in Dox-treated mice, and this was further reduced in Cav-1 KO mice compared to the wild type. To quantify lung metastasis, we explanted the mice’s lungs and plated dissociated single cells in complete RPMI media supplemented with 6-thioguanine (6-GT). The number of 6-TG-resistant 4T1 metastatic cells was significantly lower in the lungs' suspension of Cav-1 KO mice treated with Dox compared to wild-type Dox-treated mice. Our findings confirm that knockout of Cav-1 increased the sensitivity of Dox treatment and inhibited breast cancer lung metastasis in Balb/c mice. Our ongoing analysis of the molecular pathways seeks to elucidate the underlying mechanism. Citation Format: Abhishek Pandit, Dhirendra Singh, Rashmi Pathak, Shilpa Thota, Joseph Francis. Deleting caveolin-1 in epithelial cells increases doxorubicin sensitivity in advance stage of breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-28-01.

Sensitized serotonin-mediated vasodilation in a mouse model of carcinoid disease

Background: Carcinoid tumors secrete vasoactive hormones such as serotonin that impact cardiovascular function. In consequence, patients with metastatic carcinoid disease are at risk for carcinoid syndrome, characterized by profound cutaneous flushing, hypotension, and syncope. Our recent work demonstrates that in patients with carcinoid metastases, higher serotonin levels are associated with lower vascular resistance. However, it is unknown if the vascular sensitivity to serotonin is affected by carcinoid disease. The receptor that mediates the potential vasodilatory effects of serotonin in carcinoid disease is also unknown. Therefore, we hypothesize that serotonin-mediated vasodilation is increased by carcinoid metastases. To test this, we assessed vascular reactivity to serotonin ex vivo in a mouse model of carcinoid disease. Methods: J:Nu nude mice (10 weeks) received intrasplenic injection of 2x107 BON1 neuroendocrine tumor cells (BON1, n=5) or PBS-vehicle (VEH, n=5), and were monitored for 10 weeks to allow the development of liver carcinoid metastases. Animals were then euthanized, and mesentery arteries were harvested. Isolated, pressurized mesentery arteries were pre-constricted with phenylephrine. The dose-response vasodilation to serotonin and acetylcholine was measured. These dose responses were repeated in the presence of 5-HT2B receptor antagonist LY272015 or nitric oxide synthase inhibitor L-NAME. Endothelium-independent vasodilation to sodium nitroprusside was also assessed. Data are mean ± SEM. Results: Mesentery arteries from BON1 mice had a greater vasodilation in response to serotonin compared with arteries from VEH mice (65±6% vs. 44±8%, p=0.04). Incubation with L-NAME partially inhibited the vasodilation to serotonin in the BON1 arteries (42±10%, p=0.04), but not in VEH arteries (30±11%, p=0.17). LY272015 did not affect the serotonin-induced vasodilation in the BON1 or VEH arteries (BON1: 52±10%, p=0.14, VEH: 49±17%, p=0.39). In contrast to serotonin, the vasodilation to acetylcholine did not differ between BON1 and VEH arteries (77±9% vs. 61±16%, p=0.19). There were also no differences in the vasodilation to sodium nitroprusside between groups (p=0.42). Arteries from BON1 mice had a trend for greater vasoconstriction in phenylephrine compared with VEH mice (51±7% vs. 37±9%, p=0.06). Conclusions: These findings demonstrate that serotonin-mediated vasodilation is sensitized in a mouse model of carcinoid disease. This is potentially accompanied by enhanced α-adrenergic-mediated vasoconstriction, which may be a compensatory mechanism in response to sensitized vasodilatory mechanisms. Western University of Health Sciences Intramural Grant (BHM). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 4779: The effects of glutamine deprivation on ovarian cancer initiation and progression

Abstract High-grade serous ovarian cancer (HGSC) is the most common subtype of ovarian cancer. Most patients with HGSC are diagnosed at advanced stage diseases. Despite the high response rate to platinum/taxane-based chemotherapy, most of these patients will develop recurrent chemoresistant disease. Recent studies showed the cancer associated fibroblasts (CAFs) in the tumor microenvironment (TME) play an important role in modulating the malignant phenotypes of HGSC. CAFs are primarily responsible for producing factors such as cytokines and growth factors to modify the TME niche to promote the malignant phenotypes of cancer cells. Recently, we have identified a previously unrecognized mechanism in which metabolism of reactive CAFs is reprogrammed through an upregulated glutamine anabolic pathway in HGSC. This dysfunctional CAF metabolism confers atypical metabolic flexibility and adaptive mechanisms in CAFs, allowing them to harness carbon and nitrogen from noncanonical sources to synthesize glutamine in nutrient-deprived conditions existing in the TME. Our preliminary data show that tumors developed from syngeneic fallopian epithelial cell-derived cancer cells injected IP into C57BL/6 mice, which started to be fed with normal diet two weeks before tumor cells injection had significantly higher tumor burden than those fed with the glutamine-deprived diet. Moreover, tumor-bearing mice treated with glutaminase inhibitor for 3 weeks demonstrated significantly lower tumor burden than those treated with the control vehicle. These findings suggest that the interplay between CAFs and HGSC cells enriches glutamine in the ovarian TME; and glutamine depletion can not only suppress ovarian tumor growth but also prevent the development of advanced progressive diseases. In addition to the direct promoting effect of glutamine on the glutamine addicted HGSC cells, increased CAF-derived glutamine can also affect the activity of other stromal cell types such as immune cells in the TME, which subsequently modulate HGSC cell growth. Imaging mass cytometry (IMC) and Mass Spectrometry Imaging (MSI) were performed on serial tissue sections of tissue microarrays generated from the abovementioned murine samples. Mice fed with a glutamine-deprived diet or treated with the glutaminase inhibitor not only have a significantly lower tumor burden but also induced dramatic changes in the cellular and molecular composition within the TME. In particular, mice fed with a glutamine-deprived diet had greater B-cell-related immune response, reduced stemness, and EMT of ovarian cancer cells, and more activated CAFs, which may result in increased stiffness of the ECM and subsequently enhance the malignant phenotype of HGSC cells. Further studies to delineate the molecular mechanisms by which spatial cellular and metabolic profiles modulate HGSC pathogenesis and progression are warranted. Citation Format: Sammy Ferri-Borgogno, Kathryn Lu, Basant T. Gamal, Chi Lam Au Yeung, Erin Seeley, Javier A. Gomez, Akshay Basi, Jared K. Burks, Olamide Animasahun, Deepak Nagrath, Samuel C. Mok. The effects of glutamine deprivation on ovarian cancer initiation and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4779.

Abstract 148: Multiomic sequencing reveals diversity in clonal landscape and genomic alterations in a lung metastatic PDX osteosarcoma model

Abstract Introduction: Osteosarcoma is a primary malignant bone tumor characterized by the production of spindle cells resulting in immature bone formation. The lung is the most frequent site of metastatic disease and relapse occurs in more than 30 % of patients. Recent results show that relapse is due to a subset of cells with different phenotypic and genetic signatures conferring advantage to drive progression and drug resistance within the intra-tumorally heterogeneous population. Thus, developing a defined in vivo model that can identify the rare genetic subpopulation and recapitulate clonal evolution is crucial. In this study, we used whole exome, barcode and RNA sequencing to characterize the landscape of genomic alterations and also track and identify the clonal subpopulations, clonal and genetic drivers of lungs metastasis. Methods: We injected barcoded PDX (OS17) cells intravenously in ten SCID mice to track the clonal subpopulation of cells that metastasize to the lungs. Metastasized lung nodules were collected following death or euthanasia. All samples collected were snap frozen in liquid nitrogen and stored at -80°C. DNA and RNA was extracted and analyzed by PCR, NGS, WES and RNA sequencing to map clonality, mutational and evolutionary profiles. Results: Four (40%) of the mice developed lung metastases with three mice having multiple metastatic lung nodules. Surgery to remove the metastatic nodules was performed 216 ± 55.5 days following tumor cell injection. Nine metastatic lung nodules from three mice were used for sequencing and analysis. A Shannon-Weaver and Jaccard similarity index show a diversity in clonal architecture between the lung nodules. A total of 15,394 somatic variants was identified including 12,547 single nucleotide variants (SNV), 4054 synonymous, 2662 missense, 28 nonsense mutations and 1639 somatic indels. The SNVs accounted for 81% of all variants followed by CNV 11 % and indels 8% respectively. Synonymous and missense mutations were the most frequently observed in all samples. Copy number loss was two-fold compared to copy number gain across all tumors. Furthermore, the deletions span longer genomic distances compared to amplifications. The mutational spectrum showed that the highest substitutions were C&amp;gt;T/G&amp;gt;A followed by T&amp;gt;C/A&amp;gt;G; the least substitutions were T&amp;gt;A/A&amp;gt;T. Also, we identified a large set of genes with genomic aberrations. These include amplifications in VEGF, RB1, RUNX, PARP4, ICAM3, EGFR, BRCA2, COLA6A1, COLA6A2, CCND3, CDKN2D etc. Conclusions: In the current model, we identified multiple tumorigenic seeding clones and the diverse set of genes mutated in osteosarcoma. Further analysis of the mutational signature resulted in the identification of potential genes that act as drivers in the lung metastatic process. Acknowledgments: Swim Across America, the Foster Foundation and the Barbara Epstein Foundation. Citation Format: Sylvester Jusu, Wendong Zhang, Zhongting Zhang, Xu Zhaohui, Sankaranarayanan Kannan, Yifei Wang, Zhou Xin, Yi Yanhua, Michael Roth, Jonathan Gill, Richard Gorlick. Multiomic sequencing reveals diversity in clonal landscape and genomic alterations in a lung metastatic PDX osteosarcoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 148.

Abstract 1790: Pyruvate carboxylase regulates tumor progression through central carbon metabolism and immunosuppression

Abstract The tumor microenvironment (TME) is characterized by metabolic reprogramming and immune evasion, essential features in tumor progression. Tumor metabolic dysregulation, particularly the high production of lactate, plays a pivotal role in suppressing antitumor immunity within the TME. Pyruvate carboxylase (PC), responsible for converting pyruvate to oxaloacetate, is a critical determinant for lung metastasis in triple-negative breast cancer (TNBC). Herein we test the hypothesis that PC-mediated regulation of central carbon metabolism controls TME immunosurrveilance. Wild-type C57BL6/J and NSG mice received orthotopic injections of E0771 or M-Wnt tumor cells with shRNA targeting PC or a scramble control. RNA from tumors underwent transcriptomic profiling using Affymetrix Clariom D microarray (n=3-6/group per experiment). Gene set enrichment analysis identified pathways enriched in each condition, including a PC-associated hypoxia signature. Immunohistochemistry with anti-CD3 antibody and full spectrum flow cytometry assessed T cell infiltration and TME immune cell composition. Untargeted metabolomics, extracellular flux analysis, and high-resolution respirometry determined how loss of PC altered cellular metabolism. We found that suppressing PC expression increased primary tumor growth (~2 fold), potently drove immunosuppression, and remodeled cellular metabolism towards lactate production. Injection of PC-suppressed cells increased regulatory T cell levels in wild type mice. Importantly, PC-suppressed cells injected into NSG mice, which lack T, B, and NK cells, demonstrated slower, rather than accelerated, tumor growth. PC-suppression reduced oxygen consumption rate (OCR), but supply of anaplerotic carbon through exogenous malate or oxaloacetate restored OCR. Conversely, both the rate of and reliance on lactate production was increased by PC suppression. Notably, loss of immunosurveillance following downregulation of PC was reversed by inhibiting lactate transport. Additionally, lower PC expression was associated with increased hypoxia in tumors, supporting in vitro evidence of PC-repression by hypoxia. In summary, hypoxia-induced PC suppression emerges as a crucial mechanism through which primary tumors hinder antitumor immunity, partly via increased lactate production. These findings underscore the significance of PC-directed tumor metabolism as an understudied nexus in the interplay between tumor progression and antitumor immunity. Citation Format: Michael F. Coleman, Eylem Kulkoyluoglu Cotul, Alexander J. Pfeil, Alyssa N. Ho, Emily N. Devericks, Muhammad H. Safdar, Hao Chen, Numair Attaar, Violet A. Kiesel, Dorothy Teegarden, Stephen D. Hursting, Michael K. Wendt. Pyruvate carboxylase regulates tumor progression through central carbon metabolism and immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1790.

Abstract 2887: Impact of ionizing radiation on the energy metabolism of normal and tumor cells in the brain

Abstract This study aims to analyze irradiation-induced metabolic alterations in glioblastoma cells and astrocytes to enhance understanding of cellular responses and facilitate the development of more effective radiotherapeutic strategies. Metabolic reprogramming of tumor cells is considered one of the hallmarks of cancer. Studies have shown that abnormal activation of oncogenes and cancer-related signaling pathways, as well as the inactivation of tumor suppressor genes can induce metabolic reprogramming. These metabolic aberrations facilitate rapid proliferation, continuous growth, invasion, metastasis, and immune evasion. Following the application of radiotherapy, the activity of several metabolic pathways significantly changes, potentially leading to the development of radioresistance. However, a differential effect of ionizing radiation on tumor and normal cells is not well understood. Hence, quantifying irradiation-induced metabolic changes in glioblastoma cells and astrocytes is of high importance. We have developed a novel approach to measure dynamic metabolite changes upon irradiation, in vivo, in situ, and in real-time. This tool combines imaging of recently developed fluorescent biosensors via 2-photon microscopy and millimeter-scaled irradiation. Murine glioblastoma cells and astrocytes were generated with stable expression of biosensors for the quantification of intracellular concentrations of various metabolites, including lactate. Cytosolic concentrations of lactate depend on the balance between glycolytic production, mitochondrial consumption of pyruvate and lactate, and the exchange with the extracellular lactate pool via monocarboxylate transporters. Many cancer cells are characterized by an excessive conversion of glucose to lactate even under normoxic conditions. We quantified this so-called Warburg Effect in glioblastoma cells and astrocytes as the ratio between basal lactate production and the lactate increase after OXPHOS inhibition and determined irradiation-induced metabolic changes on the single-cell level. Complementarily, we used metabolomics and Seahorse analyses before and after irradiation. Our data indicate that irradiation increases oxygen consumption in glioblastoma cells but not in astrocytes. Also, we established an orthotopic glioblastoma mouse model via injection of sensor-expressing tumor cells for in vivo measurements of metabolite changes during and after irradiation. These data are expected to significantly advance the understanding of the cellular response to irradiation on a molecular level in situ and in real-time. This knowledge will help to develop combination treatments and increase the efficiency of radiotherapy. Citation Format: Marvin Kreuzer, Pascal Imseng, Bruno Weber, Martin Pruschy. Impact of ionizing radiation on the energy metabolism of normal and tumor cells in the brain [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2887.

Abstract 4137: Feasibility of a novel image guided injection tool for non-surgical implantation of orthotopic tumors

Abstract The purpose of this study was to evaluate a novel device for non-surgical implantation of tumor cells using ultrasonic (US) image guidance. There are a variety of mouse model cancer types (e.g. sub-cu, orthotopic, genetically engineered). Orthotopic tumors are increasingly prevalent but usually require surgery, which reduces throughput and injures the surrounding anatomy. Injections are an alternative to surgery, but usually require image guidance or extensive training. US guided injection systems exist, but are suboptimal because they require significant training, as the US beam is not necessarily coplanar with needle location or trajectory, reducing accuracy and throughput. We tested a new approach to image guided injection which ensures that the US beam is coplanar with the needle. To test the efficacy of the image guided injection device across a variety of common orthotopic tumors, we delivered luc-tagged cells to the prostate (2.5e5 cells in 40 µL, PC3, N=8), liver (2e6 cells in 100 µL, Hep G2, N=5), and brain (1.75e5 cells in 100 µL, U87-MG, N=3). The brain tumor model was introduced via intracardiac injection to simulate metastases. Between 3 and 8 mice were injected for each tumor model. For injection, animals were anesthetized and positioned on motion stage. A 1 mL syringe was mounted to the adjustable injection gantry with the needle co-planar with the US beam, and the target organ was visualized with US. Cells were delivered to the targets using the US live stream as a guide. Between 1 to 3 weeks after the injections, tumors were confirmed via BLI with an IVIS (Revvity, Waltham, MA, USA). For each of the three tumor cell lines, animal preparation and injections could be performed in 10 mins by recently-trained novice users. All three cell lines could be effectively delivered to the orthotopic host tissues, and this was confirmed by using BLI of the cells. This study represents the first time a co-planar US beam and needle delivery mechanism was used for the purpose of non-surgical implantation of orthotopic tumors. Future developments of this platform could include improvements to automation of needle placement and trajectory to ensure successful payload delivery by novice users. Citation Format: Thomas M. Kierski, Juan D. Rojas, Nathan Beaumont, Theresa Charity, Robert D. Marek, Kenneth H. Young, Gregory M. Palmer, Ryan C. Gessner, Tomek J. Czernuszewicz. Feasibility of a novel image guided injection tool for non-surgical implantation of orthotopic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4137.

Abstract 2189: Tirzepatide mitigates obesity-associated metabolic dysregulation and tumor progression in a mouse model of triple-negative breast cancer

Abstract Obesity is an established risk factor for breast cancer. Dietary and surgical weight loss interventions reduce breast cancer risk but have significant limitations to their widespread use as anticancer strategies. Incretin-based pharmaceuticals safely and effectively promote weight loss, but whether these drugs exert similar protective effects against cancer is unknown. Here we test whether weight loss induced by the glucagon-like peptide 1 (GLP-1)/glucose-dependent insulinotropic polypeptide (GIP) receptor agonist tirzepatide limits tumor growth in an orthotopic mouse model of triple-negative breast cancer. Ten-week-old female C57BL/6NCrl mice were randomized to either a 10 kcal% low-fat diet (control; n=14) or 60 kcal% high-fat diet to promote diet-induced obesity (DIO; n=43). After 18 weeks on diet, DIO mice were randomized by body weight to either i) remain on the high-fat diet (DIO; n=15); ii) lose weight with tirzepatide (TZP; n=13); or iii) lose weight with 30% daily calorie restriction relative to control mice (CR; n=15). All mice were subcutaneously injected with either TZP or vehicle every other day for 13 weeks with the following escalating dosing schedule: 3 nmol/kg TZP in week 1; 10 nmol/kg TZP in weeks 2-3; 20 nmol/kg TZP in weeks 4-7; 30 nmol/kg TZP in weeks 8-11; and 40 nmol/kg TZP in weeks 12-13. Fasting blood glucose and body composition was assessed after 9 weeks of weight loss, and 35,000 E0771 cells were orthotopically transplanted into the 4th mammary fat pad. Tumors growth was monitored for 4 weeks. TZP-treated mice lost on average &amp;gt;20% weight loss by week 4 and remained at or below 20% weight loss for the remainder of the study. Directly prior to E0771 tumor cell injections, TZP-treated mice had lost 23.6±8.9% body weight, versus 39.7±3.6% in CR mice. Relative to DIO mice, blood glucose control was restored in both weight loss groups (DIO: 149.5±18.5 mg/dl; TZP: 124.5±15.5 mg/dl; CR: 107.6±18.8 mg/dl) and &amp;gt;87.5% of the lost weight was attributed to fat mass, rather than lean mass, for TZP-treated and CR mice. Notably, tirzepatide suppressed tumor growth relative to DIO, but the CR group had a greater anticancer effect than TZP. Reduced tumor mass in both weight loss groups was accompanied by a significant reduction in circulating IGF-1, insulin, and leptin relative to DIO mice. These findings are timely and important because i) obesity is a highly prevalent metabolic disease that increases risk and progression of breast cancer and &amp;gt;13 other cancers; ii) incretin therapies, which show promise as highly effective weight loss drugs, are increasingly being prescribed; and iii) the tumor suppressive effects of tirzepatide described herein newly suggest incretin agonism as another propitious option for breaking the obesity-cancer link. Citation Format: Elaine M. Glenny, Alyssa N. Ho, Violet A. Kiesel, Fangxin Chen, Claire E. Gates, Evan M. Paules, Ruihan Xu, C. Alex Holt, Michael F. Coleman, Stephen D. Hursting. Tirzepatide mitigates obesity-associated metabolic dysregulation and tumor progression in a mouse model of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2189.

Abstract 2188: Tirzepatide treatment restores antitumor immunity in a model of obesity-driven cancer

Abstract Obesity is a widely and increasingly prevalent disease and a risk factor for several cancers. Recent work has shown that weight loss following bariatric surgery is potently protective against obesity-associated cancers. However, little is known about the potential anticancer effects of weight loss achieved through incretin-based therapies. We therefore interrogated the effects of the dual glucagon-like peptide 1 and glucose-dependent insulinotropic polypeptide receptor agonist tirzepatide (TZP) on body weight and tumor progression in a mouse model of obesity and transplanted colon cancer cells. Moreover, we tested whether TZP-mediated weight loss can reverse obesity-associated suppression of CD8+ T cell immunity. Male C57BL/6J mice were fed either 60 kcal% fat diet (D12492) or a sucrose-matched 10 kcal% fat diet (D12450J) for ≥15 weeks to generate diet induced obese (DIO) or control mice, respectively. Once DIO mice reached a body weight of 47 g they were randomized to begin TZP treatment or receive a vehicle control every other day for 3 weeks (when stable body weights were reached for TZP mice). All mice then received subcutaneous injections with 2.5 × 105 MC38 cells and continued to receive TZP or vehicle. To determine the role of CD8+ T cell mediated tumor control in driving the effects of TZP treatment, mice received either CD8-depleting antibodies or an isotype control at days -2, 0, 2 relative to tumor cell injection and subsequently every 4 days. Tumor mass was determined by digital scale. Fasting blood glucose, body weight loss, and body composition were monitored to determine the metabolic effects of TZP. Immune cell populations were measured by full spectrum flow cytometry. We found TZP reduced body weight, fasting blood glucose, and adiposity relative to DIO mice, as was expected. These metabolic improvements were accompanied by reduction of tumor mass in TZP mice relative to DIO mice. DIO mice had increased levels of exhausted T cells which were normalized by TZP treatment. Depletion of CD8+ cells did not alter the tumor mass of DIO mice, indicating negligible CD8+ T cell-mediated tumor control. In contrast, CD8+ cell depletion in control and TZP mice increased in tumor masses to the levels comparable to those of DIO mice. These results demonstrate that TZP can mitigate obesity-associated acceleration of tumor growth and restore markers of effective antitumor T cell immunity. Critically, we find that restoration of CD8+ T cell function is essential for TZP’s ability to limit tumor growth in DIO mice. Taken together, these results demonstrate that the metabolic improvements achieved by TZP treatment are accompanied by mitigation of obesity-driven immunosuppression. These findings shed important light on the potential for TZP and other incretin-based therapies to limit obesity-associated cancer progression, and are especially timely given the growing population of patients utilizing incretin-based therapies. Citation Format: Michael F. Coleman, Elaine M. Glenny, Tori L. McFarlane, Hannah M. Malian, Claire E. Gates, Fangxin Chen, Alyssa N. Ho, Violet A. Kiesel, Stephen D. Hursting. Tirzepatide treatment restores antitumor immunity in a model of obesity-driven cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2188.

Abstract 1496: The role of endothelial ACKR1 in breast cancer metastasis

Abstract Background: Tumor cells can co-opt mechanisms of leukocyte extravasation. Atypical Chemokine Receptor 1 (ACKR1) expression in endothelial cells has an established role in promoting leukocyte extravasation; however, the potential for endothelial ACKR1 to facilitate the metastatic process through the extravasation of tumor cells has not been studied. Methods: To understand ACKR1 function, we investigated endothelial ACKR1 expression at the distant metastatic site of the lung, a common site of breast cancer metastasis. We analyzed lung endothelial ACKR1 expression using immunofluorescent staining of lung vasculature during tumor progression in immunocompetent mice bearing orthotopically implanted E0771.LMB breast tumor cells. We assessed the timing of metastatic tumor cell arrival in the lung and evaluated the spatial relationship between disseminated tumor cells and ACKR1-positive vessels. To investigate secreted tumor factors that may mediate ACKR1 regulation in the lung, we performed intratracheal injections of TNF-alpha. We developed an endothelial cell specific ACKR1 conditional knockout mouse model (ACKR1 ECKO) to understand the impact of endothelial ACKR1 on primary tumor growth and subsequent lung metastasis in breast cancer. Results: Our results reveal a significant upregulation of ACKR1 in the lung endothelium of mice implanted with breast cancer cells in the mammary fat pad. Tumor-dependent ACKR1 expression was specifically localized to pulmonary venule endothelium, a common site of leukocyte and tumor cell extravasation. Lung endothelial ACKR1 expression became evident by day 13 after implantation, persisting through endpoint at day 28. Notably, ACKR1 upregulation occurred prior to tumor cell arrival in lungs of tumor bearing mice. We observed spatial clustering of disseminated tumor cells in proximity to ACKR1-positive vessels, implying a preferential site of tumor cell extravasation. In the absence of primary tumors, intratracheal delivery of TNF-alpha induced ACKR1 expression within lungs. When tumors were orthotopically implanted into our ACKR1 ECKO mouse model, we observed a marked decrease in lung metastases compared to control mice. Conclusion/Future Directions: We have determined that pulmonary ACKR1 expression is induced by breast tumors, and vessels expressing ACKR1 appear to act as entry points for lung metastasis. Loss of endothelial ACKR1 reduced metastasis, suggesting that endothelial ACKR1 regulates tumor cell extravasation at distant metastatic sites. We will upregulate lung endothelial ACKR1 expression and directly test its requirement for extravasation using an intravenous tumor cell injection metastasis model. Citation Format: Samuel Tanner Roach, Rishi Patel, Serena Thomas, Chinwe Ewenighi-Amankwah, Joseph Dufraine, L A. Naiche, Jan Kitajewski. The role of endothelial ACKR1 in breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1496.

Abstract 6433: Anti-CD36 monoclonal antibody can sensitize the neoadjuvant chemotherapy response in PDAC by eliminating the senescent tertiary lymphatic structure

Abstract Objective: Pancreatic ductal adenocarcinoma (PDAC) is deadly and aggressive. Neoadjuvant chemotherapy (NAC) may reduce tumor volume and alter the resectability of the tumor. The efficacy of neoadjuvant chemotherapy in patients is still limited. Tertiary lymphoid structures (TLSs) develop in peripheral non-lymphoid tissues and are essential for the immune response and correlate with chemotherapy in various tumor types. However, more research is needed to determine how TLSs affect neoadjuvant chemotherapy in PDAC. We aim to determine how neoadjuvant chemotherapy affects tumor-infiltrating lymphocytes (TLSs) in PDAC patients. We also want to find a biomarker to boost PDAC neoadjuvant chemotherapy response. Methods: We retrospectively collected formalin-fixed paraffin embedded (FFPE) tissues from 115 PDAC patients without preoperative treatment and 72 patients pretreated with neoadjuvant chemotherapy. Multispectral images were processed using AI-powered image analysis software (Tissue Gnostics; Zeiss, Germany). The 10X Genomics platform was used to sequence single cells in tissue samples from five patients who received neoadjuvant chemotherapy (NAC) and five patients who had not received any treatment before analysis (pretreatment naïve, PN). TLS was separated using Laser Microdissection. The mouse model was created by orthotopically injecting tumor cells into the pancreas. Results: Patients who received NAC have a senescent microenvironment. High-throughput single-cell sequencing showed that the senescent gene signature score was enriched in the NAC group, especially in immune cells. The percentage of p16+ CD20+ B cells and P16+ CD3+ T cells in TLSs also increased in the NAC group. Spatial transcriptomics analysis exhibited that CD36 is highly expressed in senescent TLSs, which suggested that CD36 may be a marker for senescent TLSs. Moreover, CD36+ TLSs induced the infiltration of MDSCs and regulatory T cells. CD36+ TLSs were an independent prognostic factor for poor overall survival (OS) (HR=0.67, p=0.002) and progression-free survival (PFS) (HR=0.71, p=0.003). A mice orthotopic xenograft model showed that an anti-CD36 monoclonal antibody eliminated senescent TLSs in pancreatic tissue. MDSCs and Tregs in the surrounding tissue also decreased. Additionally, in vivo KPC mice model showed that the combination of an anti-CD36 and chemotherapy reduced tumor volume and increased survival compared to chemotherapy alone. Conclusion: NAC has the potential to induce the senescence of tumor-associated TLSs, a phenomenon that has been linked to unfavorable prognosis in PDAC. CD36 serves as a viable marker for senescent TLSs that can be targeted. The administration of an anti-CD36 monoclonal antibody can potentially enhance the efficacy of chemotherapy in PDAC by selectively eliminating CD36+ senescent TLSs in mouse models. Citation Format: Jingrui Yan, Tianxing Zhou, Chao Yang, Yongjie Xie, Jun Yu, Jihui Hao. Anti-CD36 monoclonal antibody can sensitize the neoadjuvant chemotherapy response in PDAC by eliminating the senescent tertiary lymphatic structure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6433.

Abstract 5082: Novel TSHR CAR T therapy for targeting oncocytic carcinoma

Abstract Oncocytic carcinoma (Hurthle cell carcinoma) has the highest incidence of metastasis among the differentiated thyroid cancers. Metastatic disease is reported at the initial diagnosis in 10-20% of patients and 34% overall. Thyroid-stimulating hormone receptor (TSHR) expression is limited to the thyroid gland. It is abundantly expressed on thyroid tumor cells, making TSHR a compelling target for advanced thyroid cancer diagnostics and therapeutics. We developed a potent anticancer strategy for treating oncocytic carcinoma using a novel thyroid hormone stimulating receptor (TSHR)- targeted (chimeric antigen receptor) CAR T cell therapy. TSHR CAR T was generated by transducing T cells derived from healthy donors. In vitro effector functions (cytotoxicity, proliferation, degranulation) were tested in TSHR+ tumor models, and subsequent in vivo studies using NSG mice were performed to assess anti-tumor activity efficacy. We observed strong anti-tumor activity against metastatic oncocytic carcinoma cell line XCT.UC1 using the TSHR CAR T therapy in vivo. Re-challenging treated mice with XCT.UC1 cells reactivated TSHR CART cells with enhanced proliferation and complete suppression of tumor growth of the newly injected tumor cells. Our work represents a promising strategy to improve treatment outcomes of patients with metastatic oncocytic carcinoma that may provide a durable response. Citation Format: Justyna J. Gleba, Aylin Alasonyalilar-Demirer, Matthew Pawlush, Claudia Manriquez-Roman, Elizabeth L. Siegler, Robert C. Smallridge, Abba Zubair, Han W. Tun, Saad S. Kenderian, John A. Copland. Novel TSHR CAR T therapy for targeting oncocytic carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5082.

Spinal sirtuin 2 attenuates bone cancer pain by deacetylating FoxO3a

Bone cancer pain (BCP) is refractory to currently used analgesics. Recently, sirtuin 2 (SIRT2) was reported to play a vital role in neuropathic pain but its role in BCP remains unknown. It was hypothesized that spinal SIRT2 attenuates BCP by deacetylating FoxO3a and suppressing oxidative stress. The mouse model of BCP established by injecting tumor cells into the intramedullary space of the femur demonstrated that spinal SIRT2 and FoxO3a were downregulated in BCP development. Intrathecal administration of LV-SIRT2 reduced pain hypersensitivity (mechanical and thermal nociception) in BCP mice. Spinal SIRT2 overexpression upregulated FoxO3a and antioxidant genes (SOD2 and catalase) and inhibited FoxO3a acetylation, phosphorylation, and ubiquitination. Moreover, intrathecal administration of SIRT2 shRNA induced pain hypersensitivity in normal mice. Spinal SIRT2 knockdown downregulated FoxO3a and antioxidant genes and increased FoxO3a acetylation, phosphorylation, and ubiquitination. In summary, spinal SIRT2 increases FoxO3a expression in BCP mice and inhibits oxidative stress by deacetylating FoxO3a and further reducing FoxO3a phosphorylation, ubiquitination, and degradation, leading to BCP relief.