Murine Tumor Cells Research Articles

Novel treatment strategy of targeting epigenetic dysregulation in pancreatic neuroendocrine tumors.

Epigenetic dysregulation is an integral step in the progression of pancreatic neuroendocrine tumors. We hypothesized that tumor suppressor repression by DNA methyltransferase 1 in pancreatic neuroendocrine tumors could be targeted with epigenetic treatment. Resected pancreatic neuroendocrine tumors from 32 patients were stained for DNA methyltransferase 1 and scored. Human (BON1) and murine (STC) pancreatic neuroendocrine tumor cells were treated with DNA methyltransferase 1 inhibitor 5-azacytidine and chemotherapeutic agents 5-fluorouracil and temozolomide. Cell proliferation assay and tumor suppressor gene analysis were performed with qRT-PCR and Clarion S microarray. Tumor measurements were compared in a murine treatment model. High DNA methyltransferase scores were associated with high Ki-67 (6.7% vs 70.6% P < .01), mitotic rate (0.0% vs 31.3%), and grade (20.0% vs 80.4%, P < .01). Treatment with 5-azacytidine and chemotherapy resulted in a reduction of cell proliferation compared to chemotherapy alone in BON1 (77.3% vs 53.1%, P < .001) and STC (73.4% vs 34.2%, P < .001). Treatment with 5-azacytidine and chemotherapy resulted in upregulation of tumor suppressors CDKN1A (7.6 rel. fold, P < .001), BRCA2 (4.3 rel. fold, P < .001), and CDH1 (6.0 rel. fold, P= .026) in BON1 and CDKN1a (14.5 rel. fold, P < .001) and CDH (17.5 rel. fold, P < .001) in STC. In microarray, 5-azacytidine drove global genetic changes in combination treatment. Invivo tumors treated with chemotherapy measured 88.6 ± 19.54 mm3 vs 52.89 ± 10.51 mm3 in those treated with combination therapy (P= .009). Epigenetic dysregulation with DNA methyltransferase 1 is associated with pancreatic neuroendocrine tumors and is a potential targetable strategy. 5-azacytidine and chemotherapy in combination can reduce cell proliferation, upregulate silenced tumor suppressor genes, and decrease invivo tumors in pancreatic neuroendocrine tumors.

Survivin (BIRC5) Peptide Vaccine in the 4T1 Murine Mammary Tumor Model: A Potential Neoadjuvant T Cell Immunotherapy for Triple Negative Breast Cancer: A Preliminary Study

A triple negative breast cancer model using the murine 4T1 tumor cell line was used to explore the efficacy of an adjuvanted survivin peptide microparticle vaccine using tumor growth as the outcome metric. We first performed tumor cell dose titration studies to determine a tumor cell dose that resulted in sufficient tumor takes but allowed multiple serial measurements of tumor volumes, yet with minimal morbidity/mortality within the study period. Later, in a second cohort of mice, the survivin peptide microparticle vaccine was administered via intraperitoneal injection at the study start with a second dose given 14 days later. An orthotopic injection of 4T1 cells into the mammary tissue was performed on the same day as the administration of the second vaccine dose. The mice were followed for up to 41 days with subcutaneous measurements of tumor volume made every 3-4 days. Vaccination with survivin peptides was associated with a peptide antigen-specific gamma interferon enzyme-linked immunosorbent spot response in the murine splenocyte population but was absent from the control microparticle group. At the end of the study, we found that vaccination with adjuvanted survivin peptide microparticles resulted in statistically significant slower primary tumor growth rates in BALB/c mice challenged with 4T1 cells relative to the control peptideless vaccination group. These studies suggest that T cell immunotherapy specifically targeting survivin might be an applicable neoadjuvant immunotherapy therapy for triple negative breast cancer. More preclinical studies and clinical trials are needed to explore this concept further.

Quantitative Imaging of Hypoxic CAIX-Positive Tumor Areas with Low Immune Cell Infiltration in Syngeneic Mouse Tumor Models.

Limited diffusion of oxygen in combination with increased oxygen consumption leads to chronic hypoxia in most solid malignancies. This scarcity of oxygen is known to induce radioresistance and leads to an immunosuppressive microenvironment. Carbonic anhydrase IX (CAIX) is an enzyme functioning as a catalyzer for acid export in hypoxic cells and is an endogenous biomarker for chronic hypoxia. The aim of this study is to develop a radiolabeled antibody that recognizes murine CAIX to visualize chronic hypoxia in syngeneic tumor models and to study the immune cell population in these hypoxic areas. An anti-mCAIX antibody (MSC3) was conjugated to diethylenetriaminepentaacetic acid (DTPA) and radiolabeled with indium-111 (111In). CAIX expression on murine tumor cells was determined using flow cytometry, and in vitro affinity of [111In]In-MSC3 was analyzed in a competitive binding assay. Ex vivo biodistribution studies were performed to determine in vivo radiotracer distribution. CAIX+ tumor fractions were determined by mCAIX microSPECT/CT, and the tumor microenvironment was analyzed using immunohistochemistry and autoradiography. We showed that [111In]In-MSC3 binds to CAIX-expressing (CAIX+) murine cells in vitro and accumulates in CAIX+ areas in vivo. We optimized the use of [111In]In-MSC3 for preclinical imaging such that it can be applied in syngeneic mouse models and showed that we can quantitatively distinguish between tumor models with varying CAIX+ fractions by ex vivo analyses and in vivo mCAIX microSPECT/CT. Analysis of the tumor microenvironment identified these CAIX+ areas as less infiltrated by immune cells. Together these data demonstrate that mCAIX microSPECT/CT is a sensitive technique to visualize hypoxic CAIX+ tumor areas that exhibit reduced infiltration of immune cells in syngeneic mouse models. In the future, this technique may enable visualization of CAIX expression before or during hypoxia-targeted or hypoxia-reducing treatments. Thereby, it will help optimize immuno- and radiotherapy efficacy in translationally relevant syngeneic mouse tumor models.

Intratumoral CD8+ Tcells with a tissue-resident memory phenotype mediate local immunity and immune checkpoint responses in breast cancer.

CD8+ tumor-infiltrating lymphocytes with a tissue-resident memory T (TRM) cell phenotype are associated with favorable prognosis in patients with triple-negative breast cancer (TNBC). However, the relative contribution of CD8+ TRM cells to anti-tumor immunity and immune checkpoint blockade efficacy in breast cancer remains unknown. Here, we show that intratumoral CD8+ Tcells in murine mammary tumors transcriptionally resemble those from TNBC patients. Phenotypic and transcriptional studies established two intratumoral sub-populations: one more enriched in markers of terminal exhaustion (TEX-like) and the other with a bona fide resident phenotype (TRM-like). Treatment with anti-PD-1 and anti-CTLA-4 therapy resulted in expansion of these intratumoral populations, with the TRM-like subset displaying significantly enhanced cytotoxic capacity. TRM-like CD8+ Tcells could also provide local immune protection against tumor rechallenge and a TRM gene signature extracted from tumor-free tissue was significantly associated with improved clinical outcomes in TNBC patients treated with checkpoint inhibitors.

Plant extract from Caesalpinia spinosa inhibits cancer-associated fibroblast-like cells generation and function in a tumor microenvironment model

Interactions in the tumor microenvironment (TME) between tumor cells and stromal cells such as cancer-associated fibroblasts (CAF) favor increased survival, progression, and transformation of cancer cells by activating mechanisms of invasion and metastasis. The design of new therapies to modulate or eliminate the CAF phenotype or functionality has been the subject of recent research including natural product-based therapies. We have previously described the generation of a standardized extract rich in polyphenols obtained from the Caesalpinia spinosa plant (P2Et), which present antitumor activities in breast cancer and melanoma models through activities that modulate the metabolism of tumor cells or induce the development of the immune response. In this work, a model of CAF generation was initially developed from the exposure of 3T3 fibroblasts to the cytokine TGFβ1. CAF-like cells generated in this way exhibited changes in the expression of Caveolin-1 and α-SMA, and alterations in glucose metabolism and redox status, typical of CAFs isolated from tumor tissues. Then, P2Et was shown to counteract in vitro-induced CAF-like cell generation, preventing caveolin-1 loss and attenuating changes in glucose uptake and redox profile. This protective effect of P2Et translates into a decrease in the functional ability of CAFs to support colony formation and migration of 4T1 murine breast cancer tumor cells. In addition to the functional interference, the P2Et extract also decreased the expression of genes associated with the epithelial-mesenchymal transition (EMT) and functional activities related to the modulation of the cancer stem cells (CSC) population. This work is an in vitro approach to evaluate natural extracts’ effect on the interaction between CAF and tumor cells in the tumor microenvironment; thus, these results open the chance to design a more profound and mechanistic analysis to explore the molecular mechanisms of P2Et multimolecular activity and extent this analysis to an in vivo perspective. In summary, we present here a standardized polymolecular natural extract that has the potential to act in the TME by interfering with CAF generation and functionality.

Abstract B014: Mutant p53-mediated CSF1/CSF1R signaling promotes tumor invasion and lung metastasis in esophageal squamous cell carcinoma

Abstract Introduction: Mutations of the tumor suppressor p53 are detected up to 80% of esophageal squamous cell carcinoma (ESCC) cases, which in turn correlate with high metastatic rates and poor prognosis. To understand the mutant p53-mediated mechanisms in promoting ESCC metastasis, we conducted RNA-Seq and cytokine array on isogenic primary and metastatic tumor cells harvested from our mouse model of esophageal cancer harboring Trp53R172H/-, in which we have identified Colony stimulating factor 1 (Csf1) to be upregulated. Our goal is to investigate the role and mediators of CSF1 signaling through its cognate receptor CSF1R by which missense p53 mutations can promote tumor invasion and lung metastasis in ESCC. Methods: We have utilized novel L2-Cre; LSL-Trp53R172H; Rosa26LSL-YFP mice and isolated tumor cells to model metastatic ESCC. We conducted ChIP-Seq analysis for p53 on tumor cells derived from lung metastases in our mouse models. Furthermore, we have genetically and pharmacologically targeted CSF1/CSF1R signaling axis to assess its role in ESCC tumor invasion and lung metastasis. In addition to studying tumor intrinsic mechanisms, we have established quantitative multiplex immunofluorescence (qmIF) and flow cytometry approaches to characterize the changes in tumor microenvironment (TME). Results And Discussion: We demonstrate that metastatic ESCC has increased Csf1 expression compared to primary tumors, and this is dependent upon p53 mutation status, which is reinforced by the TCGA data and patient-derived tissue microarrays (TMAs). The overlay of the RNA-Seq with the ChIP-Seq analysis indicates that Csf1 is a direct gene target of p53-R172H with enriched binding motifs. Furthermore, based on the proximity ligation assay (PLA) with murine ESCC tumor cells, bromodomain and extra-terminal motif (BET) protein BRD4 interacts with p53-R172H to induce Csf1 expression. We show that the BRD4-CSF1 axis fosters tumor invasion, subcutaneous tumor growth and metastatic burden in a mutant p53 background. In accordance, upon inhibiting this signaling pathway in the tail-vein injection lung metastasis models, we have identified reduced expression of CD31+ cells and decreased infiltration of F4/80+CD163+ and F4/80+CD206+ M2-polarized macrophages at the metastatic tumor sites, indicating that the CSF1/CSF1R pathway plays a critical role in shaping the pro-metastatic and immunosuppressive TME. Finally, analysis of the esophageal cancer datasets reveals that specific p53 mutations are associated with markedly differential overall survival rates and Csf1 expression. Based upon these results, we are generating human and murine esophageal cells with distinct DNA contact and conformational p53 mutations via base editing. Conclusion: We have demonstrated novel roles and mechanisms of mutant p53-dependent CSF1/CSF1R signaling pathway in fostering ESCC tumor invasion and lung metastasis that may be applicable to other squamous cell cancers. We believe this can open up new avenues for therapeutic applications. Citation Format: Gizem Efe, Qiaosi Tang, Katherine M. Cunningham, Kensuke Sugiura, Karen Dunbar, Kausik Regunath, Kensuke Suzuki, Andres J. Klein-Szanto, Lois Resnick-Silverman, James J. Manfredi, Carol L. Prives, Anil K. Rustgi. Mutant p53-mediated CSF1/CSF1R signaling promotes tumor invasion and lung metastasis in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B014.

Macrophage regulation of Wnt pathway in canine and murine mammary cancer cells

Interactions between various cell types in a tumor microenvironment (TME) are important for cancer progression and metastasis. Tumor-associated macrophages (TAMs) play a key role in this intratumoral dialogue regulating Wnt signaling pathway in cancer cells. Co-culture of canine and murine tumor cells with TAMs lead to inhibition of the canonical Wnt pathway and activation of the non-canonical Wnt pathway in tumor cells resulting in change in tumor cell phenotype and subsequent higher potential to invasion and metastasis. These molecular changes in cancer cells that are induced by TAMs resemble the quercetin activity. This natural product is a plant flavonoid from the group of polyphenols. Its biological function is inhibition of the canonical Wnt pathway. As quercetin supplementation is broadly discussed in cancer patients, findings of this study show that this compound should be used with caution, as it may enhance cancer spread.

Galectin-9 blockade synergizes with ATM inhibition to induce potent anti-tumor immunity.

Although current cancer immunotherapies that target PD-1/PD-L1 immune checkpoint to reinvigorate exhausted T cells have achieved impressive clinical outcomes, only a small proportion of patients respond. New therapeutic targets are therefore needed to be identified to further unleash the anti-tumor potential of T cells and benefit more patients. Galectin-9 (Gal-9), initially identified as a ligand for TIM-3 to induce T cell death, acts as an immunosuppressive regulator in the tumor microenvironment (TME) but its potential as a therapeutic target remains largely elusive. Here we show that antibody neutralization of Gal-9, in combination with inhibition of Ataxia telangiectasia mutated (ATM), a kinase essential for DNA damage response (DDR), is a promising modality for cancer immunotherapy. Genetic depletion of ATM in tumors markedly potentiated anti-Gal-9 therapy in a syngeneic mouse model. Mechanistically, ATM inhibition greatly upregulated Gal-9 expression and secretion in a variety of human and murine tumor cells via the cGAS-STING-interferon β (IFNβ) innate immune pathway. Combination of Gal-9 inhibition with AZD1390, a selective ATM inhibitor currently evaluated in clinical trials, significantly suppressed tumor growth and prolonged survival in multiple syngeneic mouse models, including the poorly-immunogenic LLC lung tumors that do not respond to PD-1/PD-L1 blockade, concomitant with increased T cell infiltration. These results reveal Gal-9 induction via STING/IFNβ signaling as an important mechanism mediating tumor immune escape that could be targeted for cancer immunotherapies, and unveil a novel anti-Gal-9-based combination strategy for cancer immunotherapies in a wide variety of malignancies, including those resistant to PD-1/PD-L1 blockade.

Dendritic cell-targeting chemokines inhibit colorectal cancer progression.

Recent progress in cancer immunotherapy has shown its promise and prompted researchers to develop novel therapeutic strategies. Dendritic cells (DCs) are professional antigen-presenting cells crucial for initiating adaptive anti-tumor immunity, therefore a promising target for cancer treatment. Here, anti-tumor activities of DC-targeting chemokines were explored in murine colorectal tumor models. The correlation of chemokine messenger RNA (mRNA) expression with DC markers was analyzed using The Cancer Genome Atlas (TCGA) dataset. Murine colorectal tumor cell lines (CT26 and MC38) stably overexpressing mouse C-C motif chemokine ligand 3 (CCL3), CCL19, CCL21, and X-C motif chemokine ligand 1 (XCL1) were established by lentiviral transduction. The effect of chemokines on tumor cell proliferation/survival was evaluated in vitro by cell counting kit-8 (CCK-8) assay and colony formation assay. Syngeneic subcutaneous tumor models were used to study the effects of these chemokines on tumor growth. Ki-67 expression in tumors was examined by immunohistochemistry. Immune cells in the tumor microenvironment (TME) and lymph nodes were analyzed by flow cytometry. Expression of the four chemokines was positively correlated with the two DC markers [integrin alpha X (ITGAX) and CLEC9A] in human colorectal tumor samples. Tumoral overexpression of DC-targeting chemokines had little or no effect on tumor cell proliferation/survival in vitro while significantly suppressing tumor growth in vivo. Fluorescence-activated cell sorting (FACS) analysis showed that CCL19, CCL21, and XCL1 boosted the ratios of DCs and T cells in CD45+ leukocytes while CCL3 increased the percentage of CD45+ leukocytes in total cells in MC38 tumor. XCL1 had an additional positive effect on antigen uptake by DCs in the TME and antigen transfer to tumor-draining lymph nodes. CCL3, CCL19, CCL21, and XCL1 exhibited potent anti-tumor activities in vivo, although they might differentially regulate immune cells in the TME and antigen transfer to lymph nodes.

Miz1 promotes KRAS-driven lung tumorigenesis by repressing the protocadherin Pcdh10

Targeting KRAS-mutated non-small-cell lung cancer (NSCLC) remains clinically challenging. Here we show that loss of function of Miz1 inhibits lung tumorigenesis in a mouse model of oncogenic KRAS-driven lung cancer. In vitro, knockout or silencing of Miz1 decreases cell proliferation, clonogenicity, migration, invasion, or anchorage-independent growth in mutant (MT) KRAS murine or human NSCLC cells but has unremarkable impact on non-tumorigenic cells or wild-type (WT) KRAS human NSCLC cells. RNA-sequencing reveals Protocadherin-10 (Pcdh10) as the top upregulated gene by Miz1 knockout in MT KRAS murine lung tumor cells. Chromatin immunoprecipitation shows Miz1 binding on the Pcdh10 promoter in MT KRAS lung tumor cells but not non-tumorigenic cells. Importantly, silencing of Pcdh10 rescues cell proliferation and clonogenicity in Miz1 knockout/knockdown MT KRAS murine or human tumor cells, and rescues allograft tumor growth of Miz1 knockout tumor cells in vivo. Miz1 is upregulated in MT KRAS lung tumor tissues compared with adjacent non-involved tissues in mice. Consistent with this, Miz1 is upregulated while Pcdh10 is downregulated in human lung adenocarcinomas (LUAD) compared with normal tissues, and high Miz1 levels or low Pcdh10 levels are associated with poor survival in lung cancer patients. Furthermore, the Miz1 signature is associated with worse survival in MT but not WT KRAS LUAD, and Pcdh10 is downregulated in MT compared to WT KRAS LUAD. Taken together, our studies implicate the Miz1/Pcdh10 axis in oncogenic KRAS-driven lung tumorigenesis.

Cyclic Depsipeptides and Linear Peptides With Cytotoxic and Antiphytopathogenic Activities From Symbiotic Bacteria ofXenorhabdus(Enterobacteriales: Morganellaceae) Genus

AbstractOn the basis of biological activities of the ethyl acetate extracts of four Xenorhabdus sp., including Xenorhabdus nematophila FUM 220, Xenorhabdus nematophila FUM 221, Xenorhabdus bovienii FUM 222, and Xenorhabdus bovienii FUM 223, X. nematophila FUM 220 was preferentially selected to track the isolation of responsible compounds. Chemical study on the ethyl acetate extract of X. nematophila isolate FUM220 which is derived from the native nematode Steinernema carpocapsae (Rhabditida: Steinernematidae), was evaluated, and eleven compounds, including xenocoumacin II (1), xenortide-396 (2), xenortide A (3), xenortide-410 (4), xenortide-449 (5), xenematide A 663 (6), rhabdopeptide-574 (7), rhabdopeptide-588 (8), rhabdopeptide-687 (9), rhabdopeptide-701 (10), and nematophin-273 (11) were characterized. In this experimental study, we surveyed the antitumoral potential of bacterial extract and bacterial metabolites to treat human breast cancer (MCF-7), human lung cancer (A549), and murine Tumor (B16) cell lines. We observed that all samples were cytotoxic, but bacterial extracts of X. nematophila FUM 220 and X. bovienii FUM 223 showed higher toxicity on mentioned cell lines. Potent cytotoxic activity was found for compounds 6 and 11 with IC50 of 6.2 μg/ml against human lung cancer A549 cell lines, too. These compounds showed moderated antibacterial activity against Xanthomonas oryzae pv. oryzae strain Xoo-IR42 (Xanthomonadales: Xanthomonadaceae) (MIC of 62.5 μg/ml) and Staphylococcus aureus strain 1112 (Bacillales: Staphylococcaceae) (MIC of 100 μg/ml). The bacterial extracts from X. bovienii FUM 222 showed strong inhibition of the growth of S. aureus strain 1112, by a minimal inhibitory concentration assay (MIC of 53.5 μg/ml). Xenorhabdus genera produce metabolites with potent cytotoxic and antibacterial activity. Single compounds can be isolated, identified, and commercialized, but various species or strains may change their anticancer or antimicrobial potential. The present study brings new clues regarding the qualified of Xenorhabdus as future peptide sources for supplying natural bioactive compounds and challenge multidrug-resistant bacteria, treat cancer, and plant diseases.

First cytogenomic characterization of murine testis tumor cell line MLTC-1

The cell line MLTC-1 was established in 1982 as a transplantable Leydig cell tumor from a C57BL/6 mouse. The cell line has already been applied in &amp;gt;100 studies: still, the only information about its genetic content is given in the first description: MLTC-1 initially had a polyploid karyotype. Here, a molecular karyotyping and multicolor banding-based molecular cytogenetic study was done to provide the first chromosomal/ (molecular) cytogenetic characterization of MLTC-1. A hexaploid karyotype with 72 to 105 chromosomes was hereby characterized. Besides polyploidy, unbalanced two- and three-way translocations, dicentrics and one neocentric derivative were identified. Also, no Y-chromosome could be detected in this clearly male derived cell line. Overall, a completely unbalanced genome is present in MLTC-1 with ~20 regions being subject to copy number losses or gains. After translating these imbalances into the human genome in a standardized way, a 40% match of imbalances with human Leydig cell tumors was evident; however, about the same rate of concordance was detectable for human spermatocytic seminomas and non-seminomas as well as testicular germ cell tumor. Accordingly, MLTC-1 is better suited as an advanced testicular germ cell tumor model in general, rather than specifically for human Leydig cell tumors.

Components with Anti-Diabetic Activity Isolated from the Leaves and Twigs of Glycosmis pentaphylla Collected in Vietnam.

A phytochemical investigation of the leaves and twigs of Glycosmis pentaphylla (Rutaceae), collected in Vietnam, yielded three new compounds named glyfuran (1), glyphyllamide (2), and glyphyllazole (3), along with twenty-five known compounds (4-28). The structures of isolates were determined by IR, MS, NMR, and UV data analyses. In the anti-diabetic activity screening, (+)-isoaltholacton (4), glycoborinine (17), 2',4'-dihydroxy-4,6'-dimethoxychalcone (24), and flavokawain A (25) simultaneously exhibited inhibition of dipeptidyl peptidase-4 (DPP4) and stimulation of the glucagon-like peptide-1 (GLP-1) secretion on the murine intestinal secretin tumor cell line (STC-1).

Potent immunomodulatory and antitumor effect of anti-CD20-IL2no-alpha tri-functional immunocytokine for cancer therapy.

The anti-CD20 antibody rituximab (RTX) has substantially improved outcomes of patients with B-cell lymphomas, although more efficient therapies are needed for refractory or relapsing lymphomas. An approach to increase the clinical effectiveness of anti-tumor therapy is the use of antibody-cytokine fusion proteins (immunocytokines (ICKs)) to deliver at the tumor site the antibody effector functions and cytokines that trigger anti-tumor activities. In particular, IL-2-based ICKs have shown significant results in preclinical studies but not in clinical trials due to the toxicity profile associated to high doses IL-2 and the undesired expansion of Tregs. To improve the efficacy of RTX therapy, we fused a murine (mIgG2a) or a human (hIgG1) version of RTX to a mutated IL-2 (no-alpha mutein), which has a disrupted affinity for the high affinity IL-2 receptor (IL-2R) to prevent the stimulation of Tregs and reduce the binding to endothelial cells expressing CD25, the α chain of high affinity IL-2R. Characterization of anti-CD20-IL2no-alpha ICKs was performed by SDS-PAGE, Western-blotting and SEC-HPLC and also by several functional in vitro techniques like T-cell proliferation assays, apoptosis, CDC and ADCC assays. The in vivo activity was assessed by using murine tumor cells expressing huCD20 in C57/Bl6 mice. Both ICKs exhibited similar in vitro specific activity of their IL2no-alpha mutein moieties and kept CD20-binding capacity. Anti-CD20-IL2no-alpha (hIgG1) retained antibody effector functions as complement-dependent cytotoxicity and enhanced direct apoptosis, NK cell activation and antibody-dependent cellular cytotoxicity relative to RTX. In addition, both ICKs demonstrated a higher antitumor efficacy than parental molecules or their combination in an EL4-huCD20 tumor model in immunocompetent mice. Anti-CD20-IL2no-alpha (hIgG1) strongly expanded NK and CD8+ T cells but not Tregs in tumor-bearing mice. These findings suggest that anti-CD20-IL2no-alpha could represent an alternative treatment for B cell lymphoma patients, mainly those refractory to RTX therapy.

Citrullinated glucose-regulated protein 78 is a candidate target for melanoma immunotherapy.

Post translational modification of proteins plays a significant role in immune recognition. In particular the modification of arginine to citrulline which is mediated by PAD enzymes is increased during cellular stress (autophagy) which permits the presentation of modified epitopes upon MHC class II molecules for recognition by CD4 T cells. Citrullination also occurs in tumour cells as a result of continuous environmental stresses and increased autophagy. We have shown in animal models the efficient stimulation of citrullinated epitope specific CD4 T cells resulting in dramatic elimination/regression of tumours. The ER chaperone glucose-regulated protein 78 (GRP78) is known to also be required for stress-induced autophagy and is directly linked to autophagosome formation. GRP78 is known to be highly expressed by many tumour types. In this study we investigate the potential of targeting citrullinated GRP78 for cancer therapy. A citrullinated GRP78 specific antibody was used to assess citrullinated GRP78 expression in murine and human tumour cells by flow cytometry. Five peptides were selected and used to vaccinate HLA transgenic mice and immune responses were characterised by ex vivo cytokine ELISpot assay. T cell repertoire in humans was assessed through proliferation assays and cytokine ELISpot assay. Citrullinated peptide was identified in murine B16 melanoma by mass spectrometry and the peptide vaccine was assessed for tumour therapy in a mouse melanoma model. We show the identification CD4 T cell responses to one citrullinated GRP78 epitope that are restricted through HLA DP*0401 and HLA-DR*0101 alleles. This peptide is detected by mass spectrometry in B16 melanoma grown in vivo and citrulline specific CD4 responses to two peptides spanning this epitope mediate efficient therapy of established B16 melanoma tumours in HHDII/DP4 (p<0.0001) transgenic mouse model. Finally, we demonstrate the existence of a repertoire of responses to the citrullinated GRP78 peptide in healthy individuals (p=0.0023) with 13/17 (76%) individuals showing a response to this peptide. We propose that citrullinated GRP78 is a candidate tumour antigen and vaccination against citrullinated GRP78 may provide a promising tumour therapy approach.

Abstract IA034: Compromised myoepithelial cell differentiation correlates with DCIS to IDC transition

Abstract DCIS, breast cancer confined to mammary ducts, shares genomic and transcriptomic signatures with IDC. However for DCIS patients, standard of care for localized breast cancer remains controversial, as treatment reduces local recurrence but does not reduce risk of distant recurrence or mortality. Further, as the risk of dying from DCIS is ~5-20 fold less than from early stage breast cancer, questions remain concerning treatment harms. The fact that treatment does not reduce DCIS death rates provides indirect evidence that DCIS-stage tumor cells can disseminate and serve as reservoirs for metastatic disease. Since presence of myoepithelium is the clinical determinate of non-invasive disease, death from DCIS also implies that myoepithelial cell loss is not required for tumor cell dissemination. Here, we investigate whether changes in myoepithelial cell differentiation, rather than overt cell loss, associate with DCIS tumor cell dissemination. Using murine DCIS models and human DCIS tissue, our group reported sequential loss of myoepithelial cell differentiation markers p63, calponin, and SMA prior to progression to IDC. In human DCIS lesions delineated into low and high risk (pure DCIS vs mixed DCIS with IDC), loss of myoepithelial p63, calponin and SMA occurred more frequently in high risk DCIS lesions. Mice transgenic for calponin knockdown in the myoepithelium were at increased risk for mammary cancer. Combined, these data implicate loss in myoepithelial cell differentiation as a mediator of DCIS progression and dissemination. In human DCIS, compromised myoepithelium correlates with microinvasion and an immune infiltrate enriched in PD1+CD8 T cells. To address the role of immune cells in myoepithelial cell barrier function, we investigated 6 different immune competent mouse models of intraductal cancer (DCIS). While loss of myoepithelial differentiation markers were evident, IDC lesions developed in the absence of DCIS. Similarly, in immune compromised hosts, only IDC lesions were detected after intraductal injection of murine tumor cells, whereas human tumor cells formed stable DCIS lesions. In vitro, murine mammary tumor cells were more motile than human lines, and when tumor cells were co-cultured with myoepithelial cells, fewer myoepithelial to myoepithelial cell contacts were observed in the presence of the mouse tumor cells. Finally, in women, we find the differentiation state of mammary myoepithelium to be modulated by reproductive state, with myoepithelium during weaning–induced gland involution (an established risk window for breast cancer progression) sharing similarities with myoepithelium of high-risk DCIS lesions. In rodents, recently weaned hosts support increased DCIS to IDC transition. In sum, these studies implicate intrinsic and extrinsic regulators of myoepithelial cells, highlight the need for immune competent mouse models of DCIS, and raise the question of whether species mismatch in xenograft models impacts cellular crosstalk between myoepithelial and tumor cells critical to our understanding of the DCIS to IDC transition. Citation Format: Sarah M. Bernhardt, Elizabeth Mitchell, Tanya Russell, Sonali Jindal, Jayasri Narasimhan, Reuben J. Hoffmann, AeSoon Benson, Pepper Schedin. Compromised myoepithelial cell differentiation correlates with DCIS to IDC transition [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr IA034.

Myeloid-derived suppressor cells (MDSCs) depletion by cabozantinib improves the efficacy of anti-HER2 antibody-based immunotherapy in a 4T1-HER2 murine breast cancer model

Clinical trials using Cabozantinib have shown promising results in metastatic breast cancer. This efficacy mainly results from removing and/or polarization of tumor-promoting myeloid cells. Nevertheless, whether such myeloid-derived suppressor cells (MDSCs) depletion can be used to improve the efficacy of anti-HER2 antibodies in early breast cancer has not been defined yet. BALB/c mice were inoculated with 4T1 and 4T1-HER2 murine tumor cell lines, and after 7days, the mice were divided into different groups. Cabozantinib was orally administrated for 15 consecutive days, and anti-HER2 monoclonal antibody (mAb) 1T0 was intraperitoneally injected twice a week. Tumor size was measured every other day. Our findings indicated that Cabozantinib combined with anti-HER2 mAb dramatically reduced tumor growth and increased tumor rejection (p=0.0001). Flow cytometry analysis showed MDSC population decreased in TME, lymph nodes, and spleens by roughly 20%, 0.8%, and 35%, respectively. Myeloid suppressive phenotype was altered through inhibition of the expression of immunosuppressive factor Arg-1. Cytokine profiling of different groups indicated that the level of INF-γ was approximately two times higher than that in the control group, and IL-17 increased compared to the control group. However, IL-4 level was significantly reduced in the groups treated with Cabozantinib. These could bring about a 10% increase in CD8+ infiltration into the tumor bed and activation of tumor-draining lymph nodes and splenic T-lymphocytes. Collectively, our data provide pre-clinical evidence for using Cabozantinib to reshape the primary TME, which can enhance the effectiveness of anti-HER2 mAb immunotherapy in primary breast cancer.

Abstract C059: Targeting tumor intrinsic p38 MAPK signaling to block IL-1α-mediated inflammatory tumor-stromal crosstalk in pancreatic cancer

Abstract Introduction: Pancreatic cancer (PDAC) remains a major therapeutic challenge due to its innate and acquired chemoresistance. Activating KRAS mutations, a hallmark of PDAC, mediate autocrine effects and crosstalk within the tumor microenvironment (TME), by inducing cytokines and chemokines that promote a pro-inflammatory and immunosuppressive stroma. We have identified KRAS-driven interleukin-1α (IL-1α) as a critical mediator of the inflammatory response due to its pleiotropic effects on cancer-associated fibroblast (CAF) activation and immune evasion, however the mechanisms that regulate IL-1α production remain poorly understood. In our efforts to identify targetable kinase pathways downstream of KRAS that are involved in IL-1α expression, we identified p38 stress-associated MAPK α (p38α MAPK) as a key regulatory pathway involved in IL-1α production in PDAC tumor cells. Methods: KRAS-mutant tumor cells were treated with pharmacologic inhibitors to pathways downstream KRAS and IL1A levels determined in response by qPCR. Mutant KRAS G12D plasmid was overexpressed in mouse embryonic fibroblasts, and IL-1α levels were determined in response by qPCR and ELISA with and without p38 inhibition. Inhibition of phosphorylated p38 MAPK was achieved pharmacologically with ARRY-614 and genetically with an shRNA lentiviral system in human and murine PDAC cell lines. ChIP-qPCR was performed on a human PDAC cell line with and without p38 MAPK inhibition. Tumor cells pre-treated with ARRY-614 were cocultured with human pancreatic stellate cells, and inflammatory CAF genes were measured by qPCR. Ptf1aCre/+;LSL-KrasG12D/+; Tgfbr2flox/flox (PKT) mice, a highly aggressive PDAC genetically engineered mouse model (GEMM), were treated daily with ARRY-614, chemotherapy, or combination therapy for downstream analysis. Results: Both pharmacologic and genetic inhibition of p38 MAPK significantly reduces IL1A transcription and protein levels in multiple human and murine PDAC tumor cell lines. Furthermore, p38 MAPK inhibition reduces binding of NF-kB to the IL1A promoter and prevents IL-1α-mediated activation of CAFs, characterized by a reduction in pro-inflammatory genes. Lastly, ARRY-614 in combination with chemotherapy significantly reduces overall tumor burden and favorably remodels the tumor microenvironment in a PDAC GEMM. Conclusions: These findings provide a compelling rationale to explore p38 MAPK inhibition in tumor cells as a novel treatment strategy to suppress IL-1α-mediated stromal activation and to combine p38 MAPK inhibition with chemotherapy to overcome therapeutic resistance through modulation of the stromal and immune microenvironment in PDAC. Citation Format: Samara P. Singh, Austin R. Dosch, Siddharth Mehra, Iago de Castro Silva, Anna Bianchi, Vanessa Tonin-Garrido, Nilesh U. Deshpande, Zhiqun Zhou, Nagaraj Nagathihalli, Jashodeep Datta, Nipun B. Merchant. Targeting tumor intrinsic p38 MAPK signaling to block IL-1α-mediated inflammatory tumor-stromal crosstalk in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C059.

IMMU-05. THE INFLUENCE OF THE KETOGENIC DIET ON THE IMMUNE TOLERANT MICROENVIRONMENT IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) represents a particularly aggressive and immune-resistant cancer. Preclinical investigations have identified the anti-tumor activity of a ketogenic diet (KD) in GBM, potentially being used as a tool to target its glycolytic phenotype. Since immune cells in the tumor have a similar reliance upon nutrients to perform their individual functions, we sought to determine if the KD influenced the immune landscape of GBM. Utilizing genetically-engineered murine GBM tumor cells orthotopically implanted in immune-competent mice, we demonstrate that KD improved survival in GBM. Immunophenotyping of tumors identified a novel role KD plays in macrophage polarization, with a paradoxical 50% increase in immune-suppressive M2-macrophages and a decrease in pro-inflammatory M1-macrophages. We recapitulated KD in vitro using a modified cell culture based on comprehensive metabolomic profiling of serum in KD-fed mice. Consistent with in vivo studies, murine macrophages cultured in these conditions skewed polarization towards the M2-phenotype with immune-suppressive properties. We went on to mechanistically link these findings to the activation of transcription factor PPARg. Although anti-tumor activity was observed in mice fed a KD, we hypothesized this parallel increase in M2 macrophage polarization tempered its potential therapeutic benefit. Colony-stimulating factor 1 (CSF-1) plays a central role in macrophage differentiation, and CSF-1R inhibition is actively being investigated as a strategy to skew their polarization towards an M1-phenotype. Therefore, we tested a combination of KD with the brain-penetrant CSF-1R inhibitor BLZ945. Consistent with our hypothesis, this combination demonstrated a striking improvement in survival (p = 0.0004), with 50% of mice achieving long-term survival ( &amp;gt; 50 days). Correlative studies confirmed the capacity of BLZ945 to normalize KD-induced increases in M2s, and the combination induced an increase of anti-tumor iNOS+M1s. Combinatorial strategies using agents designed to modulate macrophage polarization represent a rational approach to improve the anti-tumor activity of KD in GBM.

PMON149 Identification of TR4 Modulators for Treatment of Cushing Disease.

Abstract Cushing Disease (CD) is caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor that causes excess adrenal-derived cortisol. It is a life-threatening "orphan disease" with an annual health care cost &amp;gt;7-fold higher than average patients. Surgical removal is the current first-line therapy but the disease frequently recurs. Repeat surgery, radiation therapy and bilateral adrenalectomy are not always successful and associated with significant morbidity. Currently available drugs or those in clinical trials for CD do not target the pituitary corticotroph tumor itself and escape from control is common with long-term use. A clear unmet need for efficacious and safe therapies that offer biochemical and tumor control exists. We hypothesize that direct targeting of corticotroph tumors to modulate ACTH and in turn, glucocorticoid secretion is the optimal way to treat CD. We recently demonstrated that the orphan testicular receptor 4 (TR4, also known as NR2C2) is a potent regulator of hypothalamic-pituitary-adrenal (HPA) axis function and directly regulates pro-opiomelanocortin (POMC) gene transcription and ACTH secretion. To identify and characterize TR4 small molecule modulators, we used the Flexi Vector system to fuse the TR4 224-615aa LBD to the 3' GAL4 DBD to generate a chimeric fusion plasmid (pBIND-TR4-LBD) that constitutively expresses a Renilla luciferase serving as an internal reporter control. We then transiently transfected our chimeric pBIND-TR4-LBD construct into human embryonic kidney (HEK293) cells which express an exogenous reporter construct pGL4.35 (GloResponse, luc2P/9XGAL4UAS-HEK293 Cell Line, Promega) that is driven by multiple copies of a GAL4 responsive upstream activating sequence (UAS). Following treatment with TR4 ligands or modulators, our TR4-LBD undergoes a conformational change and recruits additional cofactors and RNA polymerase II to the complex to drive GAL4-directed firefly luciferase expression. This dual-luciferase format allows us to detect compound cytotoxicity or off-target change in expression during chemical screening, greatly improving data quality. In a pilot drug screen of a LOPAC library and using robust z-score statistics (z-score&amp;gt;3), representing a luciferase to renilla induction &amp;gt; 3 standard deviations of baseline variability, we have identified several G-protein coupled ion channel regulators as potential "hit" TR4 modulators. These identified GPCRs regulate phosphatidylinositol and cyclic AMP effector systems, and activate several signaling pathway kinases. Mode-of-action and dose-response evaluation to calculate EC50 of these potential TR4 hit compounds is under investigation and lead compounds will be further validated using a POMC transactivation assay in murine and human corticotroph tumor cells. Our studies have begun to identify and rigorously validate compounds that efficiently and specifically abrogate TR4 actions to inhibit ACTH secretion and may lead to the discovery of safe and efficacious lead TR4 inhibitory compounds that can be further advanced as potential drug therapies for CD. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.