A Critical Reassessment of Tumor Metastasis Simulation in Murine Models: Insights Into Methodological Advances and Biological Relevance

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity. Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7. Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Background: Colorectal cancer (CRC) is the second leading cause of cancer related death. In approximately half of all CRC cases, oncogenic KRAS mutations underlie poor prognosis and drug resistance. A previous multi-genomic analysis of 4,725 biological processes with 106 human non-small-cell lung cancer cells identified nuclear transport machinery as the sole process selectively required for the survival of KRAS mutant cells. Selinexor is a clinically approved oral inhibitor of nuclear export protein Exportin 1 (XPO1/CRM1). In preclinical studies, selinexor demonstrated robust synthetical lethality with native or engineered oncogenic KRAS both in vitro and in vivo. Clinical activity of selinexor monotherapy was observed in CRC patients with various KRAS oncogenic mutations (Razak 2016 JCO). We performed preclinical studies to evaluate the effectiveness of selinexor and the anti-PD-1 antibody RMP1-14 in syngeneic mouse models of KRAS mutant CRC. Methods: Two syngeneic CT-26 KRAS mutant CRC mouse models were tested: 1) metastasis model with intra-hepatically injected CT-26 cells expressing luciferase (8 mice per group); and 2) subcutaneous model with CT-26 injected into the left flank (10 mice per group). Mice were allocated to four groups: vehicle control, selinexor (15 mg/kg, PO, once weekly), RMP1-14 (300 µg/mouse, IP, once weekly), or the combination of selinexor and RMP1-14. Tumor progression and body weight of test mice were monitored throughout the experiment. Tumor samples and plasma were collected at the end of the study for further analysis. Results: Both selinexor and RMP1-14 demonstrated single agent activity against KRAS mutant CRC in the two mouse models relative to controls. In the metastasis model, tumor growth inhibition (TGI) at Day 21 was 63.5% (p=0.0001) for selinexor and 68.9% (p<0.0001) for RMP1-14. The combination of selinexor and RMP1-14 significantly inhibited tumor growth and metastasis with a TGI of 98.9% (p<0.0001). No tumors were detected in two mice from the combination group at the end of the study. A similar trend was observed in the subcutaneous xenograft models, at Day 17, TGIs for selinexor, RMP1-14 and combination treatment were 17.6% (p=0.0921), 26.9% (p=0.0059) and 70.4% (p<0.0001), respectively. Conclusions: Synergistic anti-cancer activity of selinexor and anti-PD-1 antibody in KRAS mutant CRC mouse models warrants further clinical investigation. Citation Format: Hua Chang, Leah Henegar, Christopher J. Walker, Trinayan Kashyap, Marie Maloof, Feng Wang, Kathleen Martyn, Shira Orr, Michael G. Kauffman, Sharon Shacham, Yosef Landesman. Selinexor synergizes with anti-PD-1 antibody and inhibits tumor growth and metastasis in syngeneic mouse models of KRAS mutant colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5519.

The most facile, reproducible, and robust in vivo models for evaluating the anticancer efficacy of photodynamic therapy (PDT) are subcutaneous xenograft models of human tumors. The accessibility and practicality of light irradiation protocols for treating subcutaneous xenograft models also increase their value as relatively rapid tools to expedite the testing of novel photosensitizers, respective formulations, and treatment regimens for PDT. This chapter summarizes the methods used in the literature to prepare various types of subcutaneous xenograft models of human cancers and syngeneic models to explore the role of PDT in immuno-oncology. This chapter also summarizes the PDT treatment protocols tested on the subcutaneous models, and the procedures used to evaluate the efficacy at the molecular, macromolecular, and host organism levels.

Exon 20 genomic insertions of both epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) are oncogenic drivers and are most commonly found in non-small cell lung cancer (NSCLC). NSCLC patients with exon 20 insertions have a worse prognosis compared to those with other activating EGFR mutations. Moreover, approximately one-third of patients with exon 20 insertion mutations develop central nervous system (CNS) metastases over the course of their disease. Unfortunately, current therapeutics lack sufficient brain exposure for treating this patient population. ORIC-114 is a brain penetrant, orally bioavailable, irreversible small molecule inhibitor designed to target exon 20 insertions in EGFR and HER2. Notably, ORIC-114 is highly selective for the EGFR family of receptors, with excellent kinome selectivity compared to other reported exon 20 inhibitors, reducing the risk of off-target kinase liabilities. The superior brain penetration and free unbound exposure of ORIC-114 in preclinical studies also differentiates it from comparator EGFR and HER2 exon 20 targeted agents. To further characterize ORIC-114, in vivo studies were undertaken to assess activity in both subcutaneous and intracranial NSCLC tumor patient-derived xenograft (PDX) models. Consistent with in vitro potency and selectivity, once daily oral administration of 3 mg/kg ORIC-114 induced robust tumor regressions with greater than 100% tumor growth inhibition in the absence of significant body weight loss in an EGFR exon 20 insertion H773_V774insNPH NSCLC PDX model. In this subcutaneous model, ORIC-114 was superior to CLN-081 in efficacy and tolerability, and superior to BDTX-189 in efficacy. To investigate whether the brain-penetrant attributes of ORIC-114 translated into antitumor activity in the CNS, we utilized an intracranial PC-9 luciferase-labeled EGFR del 19 mutant cell line model. Once daily oral administration of ORIC-114 significantly regressed established intracranial NSCLC tumors and demonstrated greater efficacy than TAK-788, commensurate with the superior brain exposure of ORIC-114. We further explored dosing regimens in this intracranial model and found that ORIC-114 demonstrated equivalent regressions at 1.5 mg/kg twice daily and 3 mg/kg once daily, and strong efficacy with 1.5 mg/kg once daily dosing. Taken together, these data confirm ORIC-114 as a potent, selective, irreversible, brain penetrant exon 20 inhibitor, and a promising therapeutic candidate, including for patients with CNS metastases. Based upon these data, ORIC-114 is entering a Phase 1/1b clinical trial in genetically defined cancers. Citation Format: Jason E. Long, Soochan Kim, Ha Yeong Kim, Dong Guk Shin, Dong Hyun Park, Robert Warne, Akash Das, Ganapati Hegde, Padmini Narayanan, Lidia Sambucetti, Brenda Chan, Xi Chen, Jae H. Chang, Paul Gibbons, Jessica Sun, Matthew Panuwat, Lori S. Friedman, Melissa R. Junttila. ORIC-114, an orally bioavailable, irreversible kinase inhibitor, has superior brain penetration and antitumor activity in subcutaneous and intracranial NSCLC models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3335.

Animal models are indispensable for the study of tumorigenesis and the development of anti-cancer drugs for human pancreatic cancer. In the present study, two orthotopic xenograft mouse models were developed. AsPC-1 human pancreatic cancer cells were stably labeled with red fluorescent protein (RFP) and injected subcutaneously into nude mice. For the orthotopic tumor mass model, the formed subcutaneous tumors were cut into blocks and implanted into the pancreas of nude mice via laparotomy. For the Matrigel™ tumor block model, solidified Matrigel containing RFP-labeled AsPC-1 cells was cut into blocks and implanted into the pancreas of nude mice. A subcutaneous tumor xenograft model was used as a control. Tumor growth and metastasis were assessed using an in vivo fluorescence imaging system. Thirty-six days after implantation, all mice from the two orthotopic xenograft models (n=20 per group) and 55% of the subcutaneous xenograft mice (n=20) developed tumors. The tumor growth rate was significantly higher in the orthotopic models than that in the subcutaneous model (P<0.01). Metastasis to organs such as the liver was observed in the orthotopic tumor models. Histological examination showed that the tumors were poorly differentiated adenocarcinomas. In conclusion, two orthotopic xenograft mouse models of human pancreatic cancer were established; these exhibited greater tumor growth and metastasis than the subcutaneous xenograft mouse model.

Background: Breast cancer and melanoma are associated with a high rate of lymphatic and hematogenous metastasis which results in high rate of cancer mortality. Inflammation and hypertension (HTN) have recently emerged as causal factors for tumor progression and anti-hypertensive agents have been shown to reduce inflammation and suppress tumor growth and metastasis. Cyclooxygenase-2 (COX-2) is upregulated in most human tumors and is a potent inducer of cancer-associated inflammation that promotes tumor angiogenesis and lymphangiogenesis. This study evaluated a novel combination of a selective COX-2 inhibitor with three antihypertensive drugs to suppress tumor growth and metastasis in preclinical xenograft models. Methods: Three anti-hypertensive drugs were evaluated in this study: i) Lisinopril [LIS], an inhibitor of angiotensin-1 converting enzyme (ACE); ii) Olmesartan medoxomil (OLM), an angiotensin II receptor blocker (ARB); and iii) Hydrochlorothiazide (HCTZ), a thiazide diuretic along with Celecoxib [CEL], a selective COX-2 inhibitor. CEL, LIS, OLM, and HCTZ were evaluated either alone or in combination for tumor growth suppression and metastatic spread in an orthotopic xenograft model of triple-negative inflammatory breast cancer/SUM149 and subcutaneous xenograft models of melanoma/MDA-MB-435, glioblastoma/U87-MG, and triple-negative SUM159. Luciferase-tagged SUM149 and MDA-MB-435 cell lines were used to determine the incidence and the burden of locoregional and systemic spread. Mice were monitored twice a week for 9-16 weeks for percent weight loss, tumor volume and survival outcome. Metastatic tumor burden and incidence was measured as luciferase expression in lymph nodes and lungs and normalized to total protein. Results: In the SUM149 model, the saline treated control had an average tumor burden of 17.6 ± 8.6 x104 RLU per mg of protein in the ipsilateral lymph nodes (ILN). Two groups, OLM alone and CEL + OLM, had a statistically significant decrease in ILN burden. Olmesartan alone had a 7.1-fold decrease in tumor burden with an average of 2.4 ± 0.6 x104 RLU per mg of protein (p-value = 0.01 by Mann-Whitney test). Similar trend was observed for LIS, but not for HCTZ. In the subcutaneous model, synergistic antitumor activity was observed with OLM (p = 0.026) at low dose but not with LIS and CEL (p = ns). At high dose, LIS, OLM, and CEL showed significant inhibition of tumor growth but no synergy. When these agents were combined with Paclitaxel in the SUM159 model, there was only additive effect. HCTZ, an antihypertensive diuretic which has no direct impact on the vascular wall had no effect on tumor growth. Conclusion: These preclinical data strongly suggest a hitherto unappreciated role of ACE/ARB in tumor growth control and support the further exploration of fixed-dose combinations of CEL with ACE/ARB in cancer, especially inflammatory breast cancer. Citation Format: Cynthia Lee, Osmond J. D'Cruz, Kevin Ng, Vuong Trieu. Celecoxib fixed-dose combination: antitumor activity on tumor growth and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4902. doi:10.1158/1538-7445.AM2017-4902

Pancreatic ductal adenocarcinoma (PDAC) is a highly fatal metastatic disease associated with robust activation of the coagulation and fibrinolytic systems. However, the potential contribution of the primary fibrinolytic protease plasminogen to PDAC disease progression has remained largely undefined. Mice bearing C57Bl/6‐derived KPC (KRasG12D, TRP53R172H) tumors displayed evidence of plasmin activity in the form of high plasmin–antiplasmin complexes and high plasmin generation potential relative to mice without tumors. Notably, plasminogen‐deficient mice (Plg‐) had significantly diminished KPC tumor growth in subcutaneous and orthotopic implantation models. Moreover, the metastatic potential of KPC cells was significantly diminished in Plg‐ mice, which was linked to reduced early adhesion and/or survival of KPC tumor cells. The reduction in primary orthotopic KPC tumor growth in Plg‐ mice was associated with increased apoptosis, reduced accumulation of pro‐tumor immune cells, and increased local proinflammatory cytokine production. Elimination of fibrin(ogen), the primary proteolytic target of plasmin, did not alter KPC primary tumor growth and resulted in only a modest reduction in metastatic potential. In contrast, deficiencies in the plasminogen receptors Plg‐RKT or S100A10 in tumor cells significantly reduced tumor growth. Plg‐RKT reduction in tumor cells, but not reduced S100A10, suppressed metastatic potential in a manner that mimicked plasminogen deficiency. Finally, tumor growth was also reduced in NSG mice subcutaneously or orthotopically implanted with patient‐derived PDAC tumor cells in which circulating plasminogen was pharmacologically reduced. Collectively, these studies suggest that plasminogen promotes PDAC tumor growth and metastatic potential, in part through engaging plasminogen receptors on tumor cells.

BackgroundCancer-initiating cell (CIC), a functionally homogeneous stem-like cell population, is resonsible for driving the tumor maintenance and metastasis, and is a source of chemotherapy and radiation-therapy resistance within tumors. Targeting CICs self-renewal has been proposed as a therapeutic goal and an effective approach to control tumor growth. BMI-1, a critical regulator of self-renewal in the maintenance of CICs, is identified as a potential target for colorectal cancer therapy.MethodsColorectal cancer stem-like cell lines HCT116 and HT29 were used for screening more than 500 synthetic compounds by sulforhodamine B (SRB) cell proliferation assay. The candidate compound was studied in vitro by SRB cell proliferation assay, western blotting, cell colony formation assay, quantitative real-time PCR, flow cytometry analysis, and transwell migration assay. Sphere formation assay and limiting dilution analysis (LDA) were performed for measuring the effect of compound on stemness properties. In vivo subcutaneous tumor growth xenograft model and liver metastasis model were performed to test the efficacy of the compound treatment. Student’s t test was applied for statistical analysis.ResultsWe report the development and characterization of a small molecule inhibitor QW24 against BMI-1. QW24 potently down-regulates BMI-1 protein level through autophagy-lysosome degradation pathway without affecting the BMI-1 mRNA level. Moreover, QW24 significantly inhibits the self-renewal of colorectal CICs in stem-like colorectal cancer cell lines, resulting in the abrogation of their proliferation and metastasis. Notably, QW24 significantly suppresses the colorectal tumor growth without obvious toxicity in the subcutaneous xenograft model, as well as decreases the tumor metastasis and increases mice survival in the liver metastasis model. Moreover, QW24 exerts a better efficiency than the previously reported BMI-1 inhibitor PTC-209.ConclusionsOur preclinical data show that QW24 exerts potent anti-tumor activity by down-regulating BMI-1 and abrogating colorectal CICs self-renewal without obvious toxicity in vivo, suggesting that QW24 could potentially be used as an effective therapeutic agent for clinical colorectal cancer treatment.

Preclinical drug efficacy and tumor microenvironment (TME) investigations often utilize humanized xenograft mouse models, yet these models typically fall short in replicating the intricate TME. We developed a humanized liver metastasis (LM) model by transplanting human peripheral blood mononuclear cells (PBMCs) and assessed it against the conventional subcutaneous (SC) xenograft model, focusing on immune cell dynamics post-transplantation and immunotherapy response. NOD-scid IL2Rgammanull(NSG) were inoculated with PBMCs to create humanized models. We induced SC and LM models using HCT116 cells, to investigate and compare the distributions and transformations of immune cell subsets, respectively. Both models were subjected to anti-PD-L1 therapy, followed by an analysis the TME analysis. The LM model demonstrated enhanced central tumor infiltration by tumor-infiltrating lymphocytes (TILs) compared to the peripheral pattern of SC model. TIL subpopulations in the LM model showed a progressive increase, contrasting with an initial rise and subsequent decline in the SC model. Post-anti-PD-L1 therapy, the LM model exhibited a significant rise in central and effector memory T cells, a response absents in the SC model. Our study highlights differential TME responses between SC and LM models and introduces a robust humanized LM model that swiftly indicates the potential efficacy of immunotherapies. These insights could streamline the preclinical evaluation of TME-targeting immunotherapeutic agents.

Background: MYCN amplification is the most frequent somatically acquired genomic alteration in neuroblastoma and is a potent oncogenic driver. Targeting MYCN therapeutically has been complicated by the challenges inherent in targeting transcription factors. Recently, several groups have shown that inhibiting bromodomain and extra-terminal (BET) proteins (BRD2, BRD3, BRD4, and BRDT) and their ability to bind to acetylated lysine residues within histone tails resulted in silencing of MYC family protein expression and demonstrated therapeutic efficacy in preclinical cancer models. We hypothesized that potent inhibition of bromodomain-chromatin interactions would show anti-tumor activity in preclinical models of neuroblastoma and that reliable biomarkers of activity could be discovered. Methods: GlaxoSmithKline compounds GSK726 and GSK762 potently inhibit BET proteins (BETi) and were used for in vitro cytotoxicity and in vivo therapeutic studies, respectively. We exposed a panel of 15 cell lines to a 4-log dose range of GSK726 to determine IC50s in a luminescence-based cell viability assay. We also measured cell cycle changes by flow cytometry and apoptosis by immunoblotting for cleaved PARP. We tested the in vivo efficacy of the GSK762 in subcutaneous xenograft models (3 sensitive and 2 resistant cell lines) and in genetically engineered neuroblastoma mouse models (overexpressing MYCN and MYCN/ALK F1174L in the neural crest). For biomarker discovery, gene expression data from the neuroblastoma cell lines generated on HuGene1.0ST expression microarrays (Affymetrix) were utilized, and data were analyzed using the Limma package in R/Bioconductor. Results: Neuroblastoma cell lines (N=15) were differentially sensitive to GSK726, with a median IC50 of 128nM and a range of 17nM to &amp;gt;10uM. In sensitive cell lines, GSK726 treatment resulted in MYCN depletion, G1 arrest within 24 hours, and apoptosis. In subcutaneous xenograft models and genetically engineered neuroblastoma mouse models, GSK762 treatment resulted in tumor growth delay. Importantly, MYCN amplification status did not fully predict sensitivity to GSK726 or GSK762. Thus, to determine additional biomarkers of sensitivity, we examined baseline gene expression data at the extremes of IC50s by comparing sensitive (N=6; IC50&amp;lt;128 nM) and resistant (N=4; IC50&amp;gt;940 nM) neuroblastoma cell lines. Univariate analysis, with an FDR &amp;lt; 0.25, revealed 6 genes (PTER, PCDHB14, RFTN1, JAK2, MYCBP, and DACH1) differentially expressed between sensitive and resistant cell lines in the MYCN amplified setting. While all 6 genes predicted BETi sensitivity in the MYCN-amplified subset of cell lines, only DACH1 expression predicted sensitivity to BET inhibition irrespective of MYCN amplification status (7.92x10-7). Moreover, DACH1 levels were heterogeneously expressed in a panel of 88 primary neuroblastoma tumors (Versteeg-88) and high expression was correlated with poor patient outcome (1.74x10-5). Conclusions: BET inhibitors demonstrated significant anti-tumor activity in a subset of neuroblastoma preclinical models. Although MYCN protein levels decreased in neuroblastoma cell lines that were sensitive to BETi, MYCN amplification status alone did not fully explain BETi sensitivity. DACH1 expression serves as a candidate biomarker for sensitivity to BET inhibition. These studies will help to optimize the clinical utility of BETi in neuroblastoma and perhaps other MYC-driven malignancies. Citation Format: Robert Schnepp, Lori Hart, Pichai Raman, Laura Danielson, Maria Gagliardi, Ryan Kinsey, Anastasia Wyce, Olena Barbash, Peter Tummino, Louis Chesler, John Maris. Defining the antitumor activity and sensitivity profiles of BET inhibitors in neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B75.

The role of MDSCs in hepatocellular carcinoma – in vivo veritas?

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies and the identification of new effective therapies for HCC is urgently needed. We have previously identified EpCAM, one of the hepatic stem/progenitor markers, as a prognostic predictor of patients who received curative hepatectomy for HCC. In this preclinical study, the effects of VB4-845, an immunotoxin targeting EpCAM, were evaluated in HCC. In vitro effects of VB4-845 on human HCC cells, the cytotoxic activity, sphere-forming ability, and expression of hepatic stem/progenitor markers were analyzed. In vivo effects of VB4-845 were evaluated using subcutaneous and orthotopic liver xenograft models. In all HCC cell lines expressing EpCAM, VB4-845 showed potent cytotoxicity and was significantly effective in combination with 5-FU (p < 0.05). Although 5-FU did not affect the sphere-forming ability and increased the populations expressing other stem/progenitor markers CD133 and CD13 (p < 0.05), VB4-845 strongly suppressed the sphere-formation and decreased the population expressing CD133 and CD13 (p < 0.0005, <0.01, respectively). In subcutaneous xenograft models, the combination of VB4-845 plus 5-FU showed significant regression of tumors compared with the control (p = 0.016). Moreover, in orthotopic liver xenograft models, the combination therapy dramatically decreased the tumor volume compared with the control (p = 0.0011). Our preclinical investigation suggests that EpCAM-targeted therapy may offer a promising and novel approach for the treatment of HCC with a poorer prognosis.

Background: Head and neck squamous cell carcinoma (HNSCC) has an increased content of reactive oxygen species (ROS). However, clinical trials of antioxidant resulted in no benefit towards HNSCC anti-cancer treatment. NADPH oxidase (NOX) family is found to be the major generator of ROS which might contribute to the failure of antioxidant therapy. For the past 10 years, NOX family have been found with a range of functions in different cancers. Of which, NOX5 is the least well-understood one with limited details of signaling roles. Purpose of the study: To investigate the role of NOX5 in HNSCC. Experimental procedures: Online databases were used to analyze the expression of NOX5 in HNSCC patients. Immunohistochemistry was performed to detect the NOX5 expression level and its co-expression with hypoxia-inducible factors (HIFs) in paraffin-embedded tumor tissues surgically resected from HNSCC patients in Hong Kong. Subcutaneous xenograft model was established using NOX5-silenced HNSCC cells to evaluate the tumor growth potential in vivo . Sulforhodamine B assay and colony formation assay were used to measure the proliferation potential of NOX5-silenced HNSCC cells in vitro . Hypoxia chamber was applied to mimic the hypoxic condition of tumor tissue in vitro . Flow cytometry with Dihydroethidium staining was performed to measure the production of superoxide when silencing or overexpressing NOX5. Superoxide scavenger TEMPOL was applied to test the signaling role of superoxide generated by NOX5. Quantitative PCR and western blot were used to measure the mRNA and protein expression level of NOX5 and HIFs in HNSCC cell lines. Results: Relative mRNA expression of NOX5 was found significantly increased in HNSCC tumor tissues comparing to normal tissues both in Oncomine and TCGA datasets. Interestingly, NOX5 expression was found higher in central areas of tumor nests with the co-expression of HIF-1α and HIF-2α in our HNSCC patients’ samples. Further, NOX5 was upregulated in HNSCC cell lines under hypoxic condition (1% oxygen). Knocking-down of NOX5 impaired HNSCC cell growth both in vivo and in vitro , and this reduction was more significant under hypoxic condition in vitro . Loss of NOX5 resulted in superoxide reduction and HIF-2α impairment but not HIF-1α. Overexpressed NOX5 induced superoxide production and HIF-2α expression, and this induction was attenuated by TEMPOL. Conclusions: Hypoxia-induced NOX5 promotes HNSCC tumor growth by regulating HIF-2α via generating superoxide. NOX5 might serve as a novel therapeutic target for treatment of HNSCC. Citation Format: Siqi Chen, Wei Gao, Jimmy Yu-Wai Chan, Thian-Sze Wong. Hypoxia-induced NADPH oxidase 5 (NOX5) promotes tumor growth of head and neck squamous cell carcinoma through HIF-2a signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2648.

Background: IMGN632 is a novel CD123-targeting antibody-drug conjugate composed of a humanized anti-CD123 antibody and a DNA-alkylating IGN payload. IMGN632 is currently in Phase I dose escalation in patients with relapsed/refractory AML or BPDCN (NCT03386513), demonstrating responses across a wide range of doses. Venetoclax, a BCL-2 inhibitor, lowers the apoptotic threshold and was recently approved for elderly AML patients in combination with azacitidine. By combining these potentially synergistic anti-leukemic mechanisms in pre-clinical AML models, the increased pro-apoptotic signaling enabled by BCL-2 inhibition would act in concert with the pro-apoptotic effects of DNA damage caused by IMGN632. Aims: The combination of IMGN632 and venetoclax was evaluated in pre-clinical AML models. Methods: The IMGN632 plus venetoclax combination was evaluated in vitro in four AML cell lines (MV4–11, MOLM-13, KG-1, EOL-1), in vivo in two subcutaneous AML xenograft models (EOL-1, KG-1), and in vivo in two AML PDX models. In subcutaneous models, tumor volume was measured, complete regressions (CRs) were noted, and endpoint was determined by either clinical observations or when tumor volume reached 1000 mm3. In PDX models, survival was measured. Results: In vitro synergy was observed in 3 of the 4 cell lines tested at doses ranging from 0.5 pM to 8 pM for IMGN632, and 0.625 nM to 20 nM for venetoclax (MV4–11, MOLM-13, EOL-1), and 1 nM to 1 μM for IMGN632, and 100 nM to 1 μM for venetoclax in the KG-1 cell line, with optimal combination indexes (CI) of 0.023 (KG-1), 0.497 (MV4–11), 0.377 (MOLM-13), and 0.67 (EOL-1), indicating strong synergy across most of the cell lines tested. In the EOL-1 model (see Figure Panel A), the combination [IMGN632 (80 μg/kg, one intravenous dose) plus venetoclax (100 mg/kg, orally, daily x28)] was highly active, resulting in 4/6 CRs and a 0% T/C (percent treatment to control of the median tumor volume, at control endpoint), compared to the corresponding inactive venetoclax (1/6 CR, 69% T/C) and active IMGN632 (2/6 CRs, 11% T/C) single agents. Importantly, the four mice treated with the combination who achieved CRs remained in remission through the end of the study at >60 days. In the IMGN632-resistant KG-1 model, the combination [IMGN632 (800 μg/kg, intravenous, weekly x3) plus venetoclax (100 mg/kg, orally, daily x28)] was active (40% T/C) while corresponding single agent IMGN632 (73% T/C) and venetoclax (81% T/C) were inactive. In a venetoclax-responsive AML PDX model (NRASm, EZH2m; see Figure Panel B), the combination [low dose IMGN632 (24 μg/kg, intravenous, weekly x3) plus venetoclax (100 mg/kg, orally, daily x28)] showed 124% ILS (% increased life span), with a median survival of 148 days. This was superior to venetoclax alone (77% ILS; median survival of 117 days) and IMGN632 alone (0% ILS; median survival of 65 days). Similarly, this same combination regimen was active (40% ILS; median survival of 108 days) in a second AML PDX model (FLT3-ITD, IDH1m), while IMGN632 (10% ILS; median survival of 85 days) and venetoclax (6% ILS; median survival of 82 days) single agents were inactive.Summary/Conclusion: The combination of IMGN632 and venetoclax demonstrated synergistic cytotoxicity in vitro and greater tumor growth inhibition and prolonged survival in vivo. Collectively, these results provide support for testing the combination of IMGN632 and venetoclax in AML patients.