Syngeneic Orthotopic Mouse Model Research Articles

Investigation of Anti-Tumor Effects of an MLK1 Inhibitor in Prostate and Pancreatic Cancers.

Simple SummaryBoth prostate and pancreatic cancers are ranked in the top five leading causes of cancer death in American. In prostate cancer, the mainstay of therapeutic approaches is inhibition of the androgen receptor; however, resistance occurs within two years. In pancreatic cancer, there is no targeted therapy available, and patients have the worst survival rate compared to all other types of cancer. We identified a novel MLK1 inhibitor (NSC14465) and demonstrated anti-tumor ability in both prostate and pancreatic cancers.It was shown that mixed lineage kinase 1 (MLK1) regulates pancreatic cancer growth; however, its role in prostate cancer remains unclear. We showed that MLK1 is a tumor marker in prostate cancer by analyzing clinical gene expression data and identified a novel MLK1 inhibitor (NSC14465) from the compound library of the National Cancer Institute (NCI) using a MLK1 protein structure. The inhibitory effects of MLK1 were validated by an in vitro kinase assay and by monitoring phosphorylation signaling, and the anti-proliferation function was shown in several prostate and pancreatic cancer cell lines. We also demonstrated anti-tumor ability and prevention of cancer-related weight loss in a syngeneic orthotopic mouse model of pancreatic cancer that mimicked the tumor growth environment in the pancreas. Our results demonstrate that the MLK1 inhibitor is an anti-tumor agent for malignant prostate and pancreatic cancers.

Local ablation of gastric cancer by reconstituted apolipoprotein B lipoparticles carrying epigenetic drugs

Epigenetic inhibitors have shown anticancer effects. Combination chemotherapy with epigenetic inhibitors has shown high effectiveness in gastric cancer clinical trials, but severe side effect and local progression are the causes of treatment failure. Therefore, we sought to develop an acidity-sensitive drug delivery system to release drugs locally to diminish unfavorable outcome of gastric cancer. In this study, we showed that, as compared with single agents, combination treatment with the demethylating agent 5′-aza-2′-deoxycytidine and HDAC inhibitors Trichostatin A or LBH589 decreased cell survival, blocked cell cycle by reducing number of S-phase cells and expression of cyclins, increased cell apoptosis by inducing expression of Bim and cleaved Caspase 3, and reexpressed tumor suppressor genes more effectively in MGCC3I cells. As a carrier, reconstituted apolipoprotein B lipoparticles (rABLs) could release drugs in acidic environments. Orally administrated embedded drugs not only showed inhibitory effects on gastric tumor growth in a syngeneic orthotopic mouse model, but also reduced the hepatic and renal toxicity. In conclusion, we have established rABL-based nanoparticles embedded epigenetic inhibitors for local treatment of gastric cancer, which have good therapeutic effects but do not cause severe side effects.

Combination Therapy of Mithramycin A and Immune Checkpoint Inhibitor for the Treatment of Colorectal Cancer in an Orthotopic Murine Model.

The axis of Programmed cell death-1 receptor (PD-1) with its ligand (PD-L1) plays a critical role in colorectal cancer (CRC) in escaping immune surveillance, and blocking this axis has been found to be effective in a subset of patients. Although blocking PD-L1 has been shown to be effective in 5–10% of patients, the majority of the cohorts show resistance to this checkpoint blockade (CB) therapy. Multiple factors assist in the growth of resistance to CB, among which T cell exhaustion and immunosuppressive effects of immune cells in the tumor microenvironment (TME) play a critical role along with other tumor intrinsic factors. We have previously shown the polyketide antibiotic, Mithramycin-A (Mit-A), an effective agent in killing cancer stem cells (CSCs) in vitro and in vivo in a subcutaneous murine model. Since TME plays a pivotal role in CB therapy, we tested the immunomodulatory efficacy of Mit-A with anti-PD-L1 mAb (αPD-L1) combination therapy in an immunocompetent MC38 syngeneic orthotopic CRC mouse model. Tumors and spleens were analyzed by flow cytometry for the distinct immune cell populations affected by the treatment, in addition to RT-PCR for tumor samples. We demonstrated the combination treatment decreases tumor growth, thus increasing the effectiveness of the CB. Mit-A in the presence of αPD-L1 significantly increased CD8+ T cell infiltration and decreased immunosuppressive granulocytic myeloid-derived suppressor cells and anti-inflammatory macrophages in the TME. Our results revealed Mit-A in combination with αPD-L1 has the potential for augmented CB therapy by turning an immunologically “cold” into “hot” TME in CRC.

Abstract 2902: Beneficial effect of vactosertib combined with nal-IRI5-FU/LV in pancreatic cancer treatment

Abstract Pancreatic cancer is one of leading causes of cancer-related mortality in the world. Treatment of pancreatic cancer is difficult due to aggressiveness, distal metastasis, and genetic and epigenetic alterations. Especially, TGF-β is highly expressed in the pancreatic cancer patients, and it leads aggressive tumor metastasis. Regulation of TGF-β signaling is essential for controlling tumor progression and metastasis because TGF-β is involved in cell proliferation, epithelial-to-mesenchymal transition, angiogenesis, metastasis, and immune responses in cancer. Therefore, inhibition of TGF-β signaling may be the key concept of effective chemotherapies for metastatic pancreatic cancer. Recently, nanoliposomal iriotecan (nal-IRI), 5-flourouracil (5-FU) and leucovorin (LV) adjuvant therapy has been applied for pancreatic cancer patients' treatment. Vactosertib, one of the leading TGF-β inhibitor, has been used for clinical trials in many chemotherapy strategies. In this study, we investigated the combination treatment effect of nal-IRI/5-FU/LV and vactosertib on pancreatic cancer in syngeneic orthotopic pancreatic mouse model. The combination treatment significantly increased survival rate compare with control or single treatment of vactosertib or nal-IRI/5-FU/LV. In the histological analysis showed that cancer cell invasion to surrounding tissues was markedly reduced in vactosertib and combination treatment groups. In the RNA-sequencing analysis of tumor RNA, the apoptotic process are positively regulated in vactosertib plus nal-IRI/5-FU/LV group. Furthermore, we found the up-regulated differentially expressed gene (DEG), CCDC80, involved in cell migration, invasion and apoptosis. We ectopically overexpressed CCDC80 on pancreatic cancer cell lines and demonstrated the tumor suppressive role of CCDC80. In conclusion, the combination treatment with vactosertib with nal-IRI/5-FU/LV improved pancreatic cancer survival by inhibiting tumor invasion and inducing apoptosis. These results indicate that combination therapy can be applied to clinical trials in pancreatic cancer patient. This study supported by KHIDI grant (HI14C2640) and National R&D Program for Cancer Control, Ministry for Health and Welfare, Republic of Korea (HA17C0037). Citation Format: Eunji Hong, Sujin Park, Akira Oshima, Jinsun Heo, Jinah Park, Jihee Lee, Kyoungwha Pang, Songee Han, Hyeyeon Park, Seong-Jin Kim. Beneficial effect of vactosertib combined with nal-IRI5-FU/LV in pancreatic cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2902.

Abstract 1785: Inhibition of the IDO-Kyn-AhR cascade optimizes virotherapy in glioblastoma and depends on CD4 T-cell activation

Abstract Immune enhancement of virotherapy by reshaping the tumor immune landscape may improve its success rates. IDO, an IFNγ inducible tryptophan catabolizing enzyme, is upregulated in glioblastoma, correlating with poor prognoses. IDO-mediated tryptophan depletion in the tumor-microenvironment decreases viral replication and diminishes cell proliferation and induces apoptosis of surrounding effector T-cells. Furthermore, kynurenine (kyn), a metabolite of tryptophan, induces T-cell differentiation into Tregs. Excess kynurenine also elicits AhR-mediated lymphocyte dysfunction and immunosuppression. The immune stimulating effect of oncolytic-virus, D24-RGDOX, clinically known as DNX-2440, triggers IFNγ production contributing to a positive IDO-Kyn-AhR feedback loop. Thus, we hypothesized that combining D24-RGDOX with IDO inhibitors will synergize to treat glioblastoma. We studied the IDO-Kyn-AhR cascade in response to D24-RGDOX infection in human and murine glioblastoma cells. Orthotopic syngeneic glioblastoma mouse models were used to determine efficacy and tumor immune effects of D24-RGDOX and IDO inhibitors as single agents or as a combination treatment. We characterized IDO and AhR in D24-RGDOX infected cancers using immunofluorescence, qRT-PCR, and flow cytometry and found increased expression of both proteins in vitro and in vivo compared to mock infected controls. D24-RGDOX also increased activity of AhR in cancer cells as indicated by an AhR responsive elements transcription assay. Furthermore, we showed that viral protein expression and viral activity was enhanced in D24-RGDOX infected cultures under IDO inhibition. To test the therapeutic efficacy of combining D24-RGDOX with IDO inhibitors, we used a murine immunocompetent glioblastoma model; the combination produced 30% more long-term survivors compared D24-RGDOX alone (P=0.03, log-rank test). Next, lymphocytic depletion studies illustrated that this anticancer effect was dependent on CD4+ T-cell activation. We observed 100% survival in the re-challenged long-term glioblastoma survivors, indicating the establishment of immune memory by the combination. Functional studies showed significant increases in anti-tumor activity of splenocytes from combination-treated mice compared to D24-RGDOX-treated mice (P=0.009, student t-test). Flow cytometry studies revealed that combination-treated mice yielded the highest levels of chronically activated T-cells and lowest levels of Tregs and MDSCs compared to D24-RGDOX alone (P≤0.05 in all populations, student t-test). This microenvironment remodeling correlated with complete tumor elimination. Altogether, D24-RGDOX activates the IDO-Kyn-AhR cascade in gliomas, identifying new targets, which when inhibited have the potential to enhance the anti-glioma effect of oncolytic-viruses by reversing tumor immunosuppression. Citation Format: Teresa T. Nguyen, Dong Ho Shin, Hong Jiang, Derek A. Wainwright, Sagar Sohoni, Sumit Gupta, Ashley Ossimetha, Frederick Lang, Marta Alonso, Candelaria Gomez-Manzano, Juan Fueyo. Inhibition of the IDO-Kyn-AhR cascade optimizes virotherapy in glioblastoma and depends on CD4 T-cell activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1785.

Abstract 311: Ponatinib loaded leukocyte-based nanoparticles for osteosarcoma treatment in sarcosphere tumor model

Abstract Osteosarcoma (OS) is the most common pediatric bone tumor with a worldwide incidence of 3.4 cases per million people annually. Unfortunately, current treatments are still not sufficient to eradicate OS, due to its ability to resist the upfront standard chemotherapy. Therefore, it is essential to identify and effectively deliver novel therapeutic regimens. Multi-tyrosine kinase inhibitors have been explored as new therapeutics for different sarcomas. Among many, Ponatinib (Pon) demonstrated very potent anti-tumor activity, however, it received a black box warning from the FDA due to significant cardiovascular side effects. Recently, our lab developed novel biomimetic nanoparticles (NPs) called Leukosomes (Leuko) capable of encapsulating and effectively releasing Pon. These NPs are synthesized from activated leukocytes, maintaining leukocytes' tropism towards inflamed endothelium. Leveraging on this technology, we aim to validate the therapeutic potential of Leuko in 3D OS tumor model (sarcospheres) and their ability to target primary murine OS model while concomitantly reducing the detrimental side effects. Given its biological relevance and ability to better recapitulate the tumor structure, a 3D tumor model was chosen to reproduce key properties, such as diffusion limitations and the cellular network of solid tumors that have significant impacts on cancer drug efficacy.In the sarcospheres model, we observed efficient penetration and internalization of NPs in both, murine (RF379, 577) and human PDX derived (PDX94, pPDX202) OS cell lines, where Leuko exhibited a 20% increase in targeting vs the control Liposome (Lipo). This difference was not detected in the control 2D model. Moreover, our data demonstrated a 2-fold increase in the Leuko cytotoxic effect in 3D compared to 2D systems. Murine OS cell viability was 20% lower in sarcospheres after treatment with the IC50 for Pon. In addition, NPs induced complete inhibition of murine sarcosphere formation in the extreme limiting dilution assay (ELDA). These findings were also confirmed using the PDX derived OS cell lines. Subsequently, an in vivo intratibial syngeneic orthotopic mouse model was utilized to determine the targeting and biodistribution of the NPs. Leuko showed an increased targeting and penetration in the tumor 1 and 3h post NPs injection as anticipated by the 3D in vitro studies. By exploiting the inflammatory conditions within the tumor, which increases the Leuko accumulation, these preliminary results advocate the translational potential of this innovative formulation as a new targeted drug delivery approach for OS. This promising platform could improve the clinical therapeutic approaches and lead to improved outcomes and reduced side effects for OS patients. Citation Format: Federica Giordano, Stefania Lenna, Riccardo Rampado, April Ewing, Gherardo Baudo, Matteo Massaro, Assaf Zinger, Enrica De Rosa, Jason T Yustein, Francesca Taraballi. Ponatinib loaded leukocyte-based nanoparticles for osteosarcoma treatment in sarcosphere tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 311.

Abstract 2339: Halting head & neck squamous cell carcinoma progression by broadly targeting glutamine metabolic pathways

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, resulting in over 14,600 deaths each year in the United States alone. HNSCC is associated with human papillomavirus (HPV) infection, and tobacco use and abusive alcohol intake. Recent revolutionary immunotherapy approaches have changed the landscape of treatment options in HNSCC. However, less than 20% of HNSCC patients respond to FDA-approved anti-PD1 immune checkpoint blockade (ICB) (pembrolizumab and nivolumab), often not leading to durable responses. This highlights the unmet need to identify novel therapeutic options and biomarkers predicting more favorable response to maximize the efficacy of immune-oncology (IO) strategies for HNSCC treatment. Glutamine is a conditionally essential amino acid for rapidly proliferating cancer cells making glutamine pathway inhibition an attractive approach for anti-cancer therapy. Treatment with the glutamine antagonist (DRP-104), which irreversibly inhibits all known enzymes involved in glutamine metabolism within the tumor, resulted in metabolically halted cell growth in vitro in a large panel of cell lines (n=8) representing the spectra of HPV- and HPV+ HNSCC, with an IC50 of 0.2-25uM, Interestingly, HNSCC cells bearing genetic alterations in PIK3CA/PTEN were significantly more sensitive to glutamine antagonism in comparison to unaltered HNSCC cells. The dependence of glutamine in HNSCC growth and the increased sensitivity of PIK3CA/PTEN aberrant cells was also observed in orosphere assays and 6 distinct HNSCC tumor xenografts in NOD SCID mice. We next investigated the efficacy of glutamine suppression in the context of an intact immune system, taking advantage of new syngeneic HNSCC model system, 4MOSC and MOC1. Specifically, we recently developed orthotopic syngeneic mouse models, 4MOSC, reflecting human tobacco-related HNSCC genomic alterations and mutational landscape (Wang et al., Nature Comm 2019), while MOC1 is a DMBA-induced mouse HNSCC cell line grown in the subcutaneous flank of mice (Judd et al. Cancer Res 2011). Monotherapy administration of DRP- 104 (1.4mg/kg) was potent as a monotherapy in all three syngeneic HNSCC models. We also observed a trend of increased sensitivity when DRP-104 was combined with anti-PD-1 therapy. Our data suggest that broad glutamine antagonism using DRP-104 has therapeutic potential in HNSCC by both dismantling cancer metabolism and enhancing the anti-tumor immune response of immune checkpoint blockade. Clinical trials of DRP-104 are currently ongoing. Citation Format: Michael Allevato, Yumi Yokoyama, Robert Wild, Silvio Gutkind. Halting head & neck squamous cell carcinoma progression by broadly targeting glutamine metabolic pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2339.

P32-specific CAR T cells with dual antitumor and antiangiogenic therapeutic potential in gliomas

Glioblastoma is considered one of the most aggressive malignancies in adult and pediatric patients. Despite decades of research no curative treatment is available and it thus remains associated with a very dismal prognosis. Although recent pre-clinical and clinical studies have demonstrated the feasibility of chimeric antigen receptors (CAR) T cell immunotherapeutic approach in glioblastoma, tumor heterogeneity and antigen loss remain among one of the most important challenges to be addressed. In this study, we identify p32/gC1qR/HABP/C1qBP to be specifically expressed on the surface of glioma cells, making it a suitable tumor associated antigen for redirected CAR T cell therapy. We generate p32 CAR T cells and find them to recognize and specifically eliminate p32 expressing glioma cells and tumor derived endothelial cells in vitro and to control tumor growth in orthotopic syngeneic and xenograft mouse models. Thus, p32 CAR T cells may serve as a therapeutic option for glioblastoma patients.

Effects of the DRD2/3 antagonist ONC201 and radiation in glioblastoma

BackgroundGlioblastoma (GBM) is the deadliest of all brain cancers in adults. The current standard-of-care is surgery followed by radiotherapy and temozolomide, leading to a median survival time of only 15 months. GBM are organized hierarchically with a small number of glioma-initiating cells (GICs), responsible for therapy resistance and tumor recurrence, suggesting that targeting GICs could improve treatment response. ONC201 is a first-in-class anti-tumor agent with clinical efficacy in some forms of high-grade gliomas. Here we test its efficacy against GBM in combination with radiation. MethodsUsing patient-derived GBM lines and mouse models of GBM we test the effects of radiation and ONC201 on GBM self-renewalin vitro and survivalin vivo.A possible resistance mechanism is investigated using RNA-Sequencing. ResultsTreatment of GBM cells with ONC201 reduced self-renewal, clonogenicity and cell viabilityin vitro. ONC201 exhibited anti-tumor effects on radioresistant GBM cells indicated by reduced self-renewal in secondary and tertiary glioma spheres. Combined treatment of ONC201 and radiation prolonged survival in syngeneic and patient-derived orthotopic xenograft mouse models of GBM. Subsequent transcriptome analyses after combined treatment revealed shifts in gene expression signatures related to quiescent GBM populations, GBM plasticity, and GBM stem cells. ConclusionsOur findings suggest that combined treatment with the DRD2/3 antagonist ONC201 and radiation improves the efficacy of radiation against GBMin vitroandin vivothrough suppression of GICs without increasing toxicity in mouse models of GBM. A clinical assessment of this novel combination therapy against GBM is further warranted.

Targeting CSF1R Alone or in Combination with PD1 in Experimental Glioma.

Simple SummaryGlioblastomas are incurable tumors of the central nervous system. Currently, treatment strategies combine neurosurgical intervention, radiation therapy, and chemotherapy. Yet, clinical experience shows that tumors acquire escape mechanisms. Furthermore, the tumor-associated microenvironment, including macrophages expressing the receptor CSF1R, promote and nourish tumor cells. The so-called PD1/PDL1 axis is a major reason why tumors can grow with a “magic hat”; i.e., unrecognized from the immune system. The aim of our study was to assess treatment strategies that target macrophages in the microenvironment by blocking CSF1R alone or in combination with PD1 blockade. Using an immune competent mouse model and an ex vivo microtumor model using freshly resected glioblastoma material, we observed prolonged survival of treated mice and an improved “attack” of the immune system. We conclude that targeting CSF1R is a promising strategy that should be explored in clinical trials, potentially in combination with PD1 blockade.Glioblastoma is an aggressive primary tumor of the central nervous system. Targeting the immunosuppressive glioblastoma-associated microenvironment is an interesting therapeutic approach. Tumor-associated macrophages represent an abundant population of tumor-infiltrating host cells with tumor-promoting features. The colony stimulating factor-1/ colony stimulating factor-1 receptor (CSF-1/CSF1R) axis plays an important role for macrophage differentiation and survival. We thus aimed at investigating the antiglioma activity of CSF1R inhibition alone or in combination with blockade of programmed death (PD) 1. We investigated combination treatments of anti-CSF1R alone or in combination with anti-PD1 antibodies in an orthotopic syngeneic glioma mouse model, evaluated post-treatment effects and assessed treatment-induced cytotoxicity in a coculture model of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TILs) ex vivo. Anti-CSF1R monotherapy increased the latency until the onset of neurological symptoms. Combinations of anti-CSF1R and anti-PD1 antibodies led to longterm survivors in vivo. Furthermore, we observed treatment-induced cytotoxicity of combined anti-CSF1R and anti-PD1 treatment in the PDM/TILs cocultures ex vivo. Our results identify CSF1R as a promising therapeutic target for glioblastoma, potentially in combination with PD1 inhibition.

Tumor-Secreted Extracellular Vesicles Regulate T-Cell Costimulation and Can Be Manipulated To Induce Tumor-Specific T-Cell Responses

Colorectal cancer is a major cause of cancer-related deaths worldwide. Immune checkpoint blockade therapies are effective in 30%-60% of the microsatellite instable-high subtype. Unfortunately, most patients with colorectal cancer (>85%) have microsatellite stable tumors that do not respond. In this study, we aimed to decipher the underlying tumor-intrinsic mechanisms critical for improving immunotherapy in colorectal cancer. We used human and mouse tumor samples, cell lines, human colorectal cancer organoids, and various syngeneic orthotopic mouse models of late-stage colorectal cancer to define the effects of tumor cell-secreted extracellular vesicles (EVs) on antitumor immune response. Our analyses of human colorectal cancer immune profiles and tumor-immune cell interactions showed that tumor-secreted EVs containing microRNA miR-424 suppressed the CD28-CD80/86 costimulatory pathway in tumor-infiltrating T cells and dendritic cells, leading to immune checkpoint blockade resistance. Modified tumor-secreted EVs with miR-424 knocked down enhanced T-cell-mediated antitumor immune response in colorectal cancer tumor models and increased the immune checkpoint blockade response. Intravenous injections of modified tumor-secreted EVs induced tumor antigen-specific immune responses and boosted the immune checkpoint blockade efficacy in colorectal cancer models that mimic aggressively progressing, late-stage disease. Collectively, we show a critical role for tumor-secreted EVs in antitumor immune regulation and immunotherapy response, which could be developed as a novel treatment for immune checkpoint blockade-resistant colorectal cancer.

High-dimensional immunotyping of tumors grown in obese and non-obese mice.

ABSTRACTObesity is a disease characterized by chronic low-grade systemic inflammation and has been causally linked to the development of 13 cancer types. Several studies have been undertaken to determine whether tumors evolving in obese environments adapt differential interactions with immune cells and whether this can be connected to disease outcome. Most of these studies have been limited to single-cell lines and tumor models and analysis of limited immune cell populations. Given the multicellular complexity of the immune system and its dysregulation in obesity, we applied high-dimensional suspension mass cytometry to investigate how obesity affects tumor immunity. We used a 36-marker immune-focused mass cytometry panel to interrogate the immune landscape of orthotopic syngeneic mouse models of pancreatic and breast cancer. Unanchored batch correction was implemented to enable simultaneous analysis of tumor cohorts to uncover the immunotypes of each cancer model and reveal remarkably model-specific immune regulation. In the E0771 breast cancer model, we demonstrate an important link to obesity with an increase in two T-cell-suppressive cell types and a decrease in CD8 T cells.

Qa-1b Modulates Resistance to Anti-PD-1 Immune Checkpoint Blockade in Tumors with Defects in Antigen Processing.

Immune checkpoint blockade (ICB) has improved cancer care, but ICB is only effective in some patients. The molecular mechanisms that influence ICB therapy response are not completely understood. The non-classical MHC class I molecule HLA-E and its mouse ortholog, Qa-1b, present a limited set of peptides in a TAP1-dependent manner to the NKG2A/CD94 heterodimer to transduce an inhibitory signal to natural killer (NK) and CD8+ T cells. However, deficiency of TAP1 allows Qa-1b to present an alternative peptidome to Qa-1b-restricted T-cell receptors of cytotoxic T cells. In this study, we used CRISPR-Cas9 to study the relationship between TAP1, Qa-1b, and response to anti-PD1 therapy. We hypothesized that immunotherapy response in TAP1-deficient tumors would be influenced by Qa-1b. Strikingly, using a syngeneic orthotopic mouse model, we found that although TAP1-deficient tumors were resistant to anti-PD1 treatment, anti-PD1 response was significantly enhanced in tumors lacking both TAP1 and Qa-1b. This increased sensitivity is partially dependent on NK cells. TAP1-deficient tumors were associated with an increase of intratumoral regulatory T cells (Treg) and neutrophils, whereas tumors lacking both TAP1 and Qa-1b exhibited an increased CD8+ T-cell to Treg ratio. These data suggest that direct inhibition of Qa-1b may alter the immune microenvironment to reverse resistance to anti-PD1 therapy, particularly in the context of antigen-processing defects. IMPLICATIONS: This study reveals important functional crosstalk between classical TAP-dependent MHC complexes and Qa-1b/HLA-E, particularly in tumors with impaired antigen-processing machinery. This can dramatically influence immunotherapy efficacy.

Abstract PR008: Identifying mechanisms of immune evasion in microsatellite instable endometrial cancer mouse models

Abstract Introduction: Microsatellite instability (MSI), caused by defects in DNA mismatch repair genes, including MLH1 and MSH2, occur in 30% of endometrial cancers (EC). High mutational loads associated with MSI have been associated with greater immunogenicity, and an increase in immune inhibitory factors such as PD-1 and PD-L1. While immunotherapy with anti-PD-L1 has shown promising results in MSI ECs, up to 40% did not respond, suggesting there are alternate immunosuppressive mechanisms. To improve understanding of response/resistance to immunotherapy, we evaluated immune activation and evasion using a novel immunocompetent mouse model of MSI EC. Methods: Endometrial lesions were evaluated in mice with MSH2 loss targeted to the endometrium (PRCre+MSH2flox/flox). When aged to 12-16 months, 22% develop hyperplasia and 18% develop spontaneous EC. MSI status was confirmed by PCR. Immune infiltration and function were assessed by immunohistochemistry (IHC) and Nanostring digital spatial profiling. Tumors were evaluated for tumor infiltrating lymphocytes (TIL) based on CD8a expression and found to separate into TILHIGH and TILLOW groups and further characterized to determine immune activation/resistance mechanisms using transcriptome analysis with Affymetrix Clariom D assay and validation by RT-PCR. A gene signature score approach was used to quantify immune-related expression changes for tumors from PRCre+MSH2flox/flox mice and MSI EC patient data from TCGA. Cell lines were generated from tumors and used to evaluate a syngeneic orthotopic mouse model for immunotherapy studies, where immune infiltration was characterized. Results: All endometrial tumors were MSI and included endometrioid, serous, and mixed histologies. Tumors showed varying degrees of CD8+ T cell infiltration, irrespective of histology. TILHIGH tumors had increased infiltration of dendritic cells and Type I interferon response, including genes IFI203 (fold-change, FC=18.5), IFI204 (FC=11.1) and CXCL9 (FC=5.7). There was no significant difference in type II interferon response. TILLOW tumors had low expression of IFN-associated genes. TILHIGH tumors also exhibited increased immune exhaustion markers such as TIM3 (FC=2.4) and LAG3 (FC=3.2). Tumoral PD-L1 expression was negative (<1% of positive cells). Similar to our mouse model, TILHIGH MSI uterine cancer patients from TCGA also showed activation of type I interferon, increased dendritic cell infiltration, and increased T cell exhaustion markers. Primary cell lines used in an orthotopic mouse model retained the immune profile of the original tumor. Conclusions: Elucidating immune activation and evasion mechanisms is critical for improving immunotherapies. The PRCre+MSH2flox/flox mouse model reflects the spectrum of immunogenicity observed in patients. A panel of primary cell lines generated from this model are an important tool that enable studies of immunotherapy efficacy and resistance mechanisms, in both immunogenic and non-immunogenic tumors. Citation Format: Brenda Melendez, Emily Hinchcliff, Nisha Gokul, Xiaoping Su, Daniel McGrail, Elizabeth Whitley, Russell Broaddus, Rosemarie Schmandt, Karen Lu, Melinda Yates. Identifying mechanisms of immune evasion in microsatellite instable endometrial cancer mouse models [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PR008.

Inflammatory microenvironment of fibrotic liver promotes hepatocellular carcinoma growth, metastasis and sorafenib resistance through STAT3 activation.

The pro‐inflammatory and pro‐fibrotic liver microenvironment facilitates hepatocarcinogenesis. However, the effects and mechanisms by which the hepatic fibroinflammatory microenvironment modulates intrahepatic hepatocellular carcinoma (HCC) progression and its response to systematic therapy remain largely unexplored. We established a syngeneic orthotopic HCC mouse model with a series of persistent liver injury induced by CCl4 gavage, which mimic the dynamic effect of hepatic pathology microenvironment on intrahepatic HCC growth and metastasis. Non‐invasive bioluminescence imaging was applied to follow tumour progression over time. The effect of the liver microenvironment modulated by hepatic injury on sorafenib resistance was investigated in vivo and in vitro. We found that the persistent liver injury facilitated HCC growth and metastasis, which was positively correlated with the degree of liver inflammation rather than the extent of liver fibrosis. The inflammatory cytokines in liver tissue were clearly increased after liver injury. The two indicated cytokines, tumour necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6), both promoted intrahepatic HCC progression via STAT3 activation. In addition, the hepatic inflammatory microenvironment contributed to sorafenib resistance through the anti‐apoptotic protein mediated by STAT3, and STAT3 inhibitor S3I‐201 significantly improved sorafenib efficacy impaired by liver inflammation. Clinically, the increased inflammation of liver tissues was accompanied with the up‐regulated STAT3 activation in HCC. Above all, we concluded that the hepatic inflammatory microenvironment promotes intrahepatic HCC growth, metastasis and sorafenib resistance through activation of STAT3.

CSIG-16. SEXUAL DIMPORHISM IN IRON ACQUISITION IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most aggressive brain cancer. Sex differences in incidence and clinical outcomes have been reported, however, our knowledge of contributing mechanisms is limited. Iron acquisition is key to robust tumor growth. Upregulation of Transferrin (Tf, iron transport protein)/Transferrin receptor (TfR) is found in multiple different cancers. We have identified H-ferritin (FTH1) as involved in iron transport and explore its uptake in GBM in this study. We interrogated iron uptake in a syngeneic orthotopic mouse model (GL261 cells) using male and female mice. After the tumors were established, radioactive 125I labeled Tf and FTH1 proteins were injected retro-orbitally in the mice. After 24 hours, tumors were removed, homogenized and analyzed for Tf and FTH1 uptake. There was a significant difference in Tf uptake into the tumor versus matched non-tumor tissue in both males and females and the uptake in the tumors was 1.5-fold higher in males than females. There was no significant difference in FTH1 uptake between male and female tumors nor between tumor and matched non-tumor brain tissue. Binding analyses were performed on homogenized samples of human male and female GBM tissue samples using 125I labeled Tf and FTH1. Tumors from males had increased binding of both proteins compared to tumors from females. We next queried the TCGA database and found in females, high TfR expression was associated with poor survival but not in males. TCGA database revealed a robust expression of Tim1, a putative receptor for FTH1, but its expression did not relate to survival. In summary, this study demonstrates FTH1 binding to GBMs and sexual dimorphism in iron acquisition via Tf and survival.

IMMU-07. TUMOR STROMA–TARGETING ANTIBODY-CYTOKINE CONJUGATES TO CONVERT THE IMMUNOLOGICALLY COLD GLIOMA MICROENVIRONMENT INTO A HOT ONE

Abstract Glioblastoma is an immunological “desert”. The administration of pro-inflammatory cytokines could shift the balance between tumor-associated immune suppression and anti-tumor immunity but the systemic administration of therapeutically active doses of pro-inflammatory cytokines is hampered by toxic side effects. We investigated different antibody-cytokine fusion products that enable a targeted delivery of interleukin (IL-) 2, IL-12 or tumor-necrosis factor (TNF)α to the tumor site upon systemic administration by binding to a tumor-specific epitope of fibronectin. We confirmed the target antigen expression in ex vivo sections of orthotopic syngeneic glioma mouse models and human glioblastoma samples and characterized the distribution of these antibody-conjugates in glioma-bearing mice upon systemic administration using in vivo imaging. Subsequently, we demonstrated potent anti-tumor activity of these antibody-fusion proteins in fully immunocompetent orthotopic mouse glioma models and characterized their mode of action. We also translated this immunotherapeutic strategy to treat patients with recurrent glioblastoma systemically with an antibody-fusion protein that enables the targeted delivery of TNFα to the tumor site. This was well tolerated, led to a treatment-associated tumor necrosis and increased the number of tumor-infiltrating T cells. This work builds a basis for future studies with antibody-cytokine fusion proteins as a promising treatment strategy for central nervous system tumors.

65P The new mouse anti-nNav1.5 monoclonal antibody

Neonatal Nav1.5 (nNav1.5) is a potent tumour metastatic marker found re-expressed in human breast cancer cells with aggressive phenotype and breast tumour tissue positive for lymph node metastasis. Mouse anti-nNav1.5 monoclonal antibody, 4H8 was recently obtained.Neonatal Nav1.5 (nNav1.5) is a potent tumour metastatic marker found re-expressed in human breast cancer cells with aggressive phenotype and breast tumour tissue positive for lymph node metastasis. Mouse anti-nNav1.5 monoclonal antibody, 4H8 was recently obtained. This study aimed to assess the specificity of the 4H8 against nNav1.5. 4H8 was used as primary antibody in Western blot and immunocytochemistry tested on cells and cell lysates, respectively of two human breast cancer cell line; MDA-MB-231+nNav1.5 and MCF-7-nNav1.5, a mouse mammary cancer cell line, 4T1+nNav1.5, a non-cancerous human breast epithelial, MCF-10A-nNav1.5 and tumour tissue lysate from an orthotopic syngeneic mouse model (4T1+nNav1.5/BALB/c). were consistent with nNav1.5 protein being more abundantly expressed in aggressive human breast cancer cell line, MDA-MB-231+nNav1.5 and mouse mammary cancer cell line, 4T1+nNav1.5 and tumour tissue lysate from an orthotopic syngeneic mouse model (4T1+nNav1.5/BALB/c) as compared to MCF-7-nNav1.5 and MCF-10A-nNav1.5. The new antibody described in this work can be a novel tool in studying nNav1.5 expression in breast cancer.

Abstract NG12: Mechanisms governing efficacy of combination CD40 agonist and anti-PD-L1 in pancreatic ductal adenocarcinoma

Abstract NG12: Mechanisms governing efficacy of combination CD40 agonist and anti-PD-L1 in pancreatic ductal adenocarcinoma

Abstract 1711: Mechanical strain induces phenotypic changes in breast cancer cells and promotes immunosuppression in the tumor microenvironment

Abstract Introduction: Breast cancer (BCa) occurs with a complex, three-dimensional microenvironment that involves heterogeneous biochemical and biophysical cues. Understanding how mechanical properties within the tumor microenvironment (TME) regulate breast cancer phenotype and immunosuppression is of great interest. Materials and Methods: BCa cells (MCF-7, MDA-MB-231 or 4T1.2) cultured to confluence on collagen coated FlexCell culture plates were subjected to 10% uniaxial cyclic/oscillatory strain at 0.3 Hz, or 10% constant strain, or no strain for 48 hours. They were isolated for analysis of proliferation (MTT assay and cell count by trypan blue), and migration (transwell and wound healing assay). Exosomes from conditioned media were isolated by differential centrifugation or using the Total Exosome Isolation kit. The purified exosomes were quantified by NanoSight and characterized by ImageStream. 5 × 105 4T1.2 cells or PKH67-labeled strained or control cells were injected into the mammary fat pad of BALB/c mice. Tumor volume was measured at the indicated time points after injection. Tumor-infiltrating immune cells and the internalization of exosomes were analyzed by flow cytometry on day 14 post implantation. In some experiments, on day 6 after tumor injection, 7.5 × 108 PHK67-labeled tumor cell-derived exosomes or PBS were injected into the tumor nodule. Tumor tissues were harvested for analysis of the internalization of exosomes by immune cells and tumor cells on days 2 and 8 after exosome injection. Results: We show that mechanical strain enhanced the proliferation and migration of BCa cells in vitro. Exosome concentrations produced by triple negative breast cancer (TNBC) cells were increased following exposure to oscillatory strain. Phenotyping exosomes by ImageStream showed that the percentages of CD81+PD-L1+ and CD63+PD-L1+ exosomes were increased after exposure to oscillatory strain. Using a syngeneic orthotopic mouse model of TNBC, we showed that preconditioning with mechanical strain increased tumor growth. The percentages of tumor-infiltrating monocytic myeloid-derived suppressor cells (M-MDSC) and recruited macrophages were increased while CD8+ T cells decreased in the TME of mice implanted with 4T1.2 cells preconditioned with oscillatory strain. Further, exosome internalizations by M-MDSC and recruited macrophages were elevated when tumor cells were preconditioned with oscillatory strain. Moreover, exosomes internalization by immune cells and tumor cells in TME were identified by PKH67 positive signals on days 2 and 8 after injection of PKH67-labeled exosomes into tumor nodules by flow cytometry analyses and confocal microscope imaging. Conclusions: Our data indicate that exposure to mechanical strain promotes invasive and pro-tumorigenic phenotypes in BCa, alters exosome production by BCa and induces immunosuppression in the TME. Citation Format: Yong Wang, Kayla F. Goliwas, Paige E. Severino, Kenneth Hough, Derek Van Vessem, Hong Wang, Sultan Tousif, Roy P. Koomullil, Andra R. Frost, Selvarangan Ponnazhagen, Joel L. Berry, Jessy S. Deshane. Mechanical strain induces phenotypic changes in breast cancer cells and promotes immunosuppression in the tumor microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1711.