Patient-derived Orthotopic Xenograft Mouse Model Research Articles

Transforming Albumin into a Trojan Horse of Immunotherapy-Resistant Colorectal Cancer with a High Microsatellite Instability.

The therapeutic response of microsatellite instability-high (MSI-H) colorectal cancer (CRC) to immune checkpoint inhibitors (ICI) is indeed surprising; however, the emergence of acquired resistance poses an even greater threat to the survival of these patients. Herein, bioinformatics analysis of MSI-H CRC samples revealed that Wnt signaling pathway represents a promising target for acquired immune reactivation, while subsequent analysis and biochemical testing substantiated the inclination of Wnt-hyperactive CRC cells to engage in macropinocytosis with human serum albumin (HSA). These findings have inspired us to develop an engineered HSA that not only possesses the ability to specifically target cancer cells but also effectively suppresses the Wnt/β-catenin cascade within these malignant cells. In pursuit of this objective, a comprehensive screening of reported Wnt small-molecule inhibitors was conducted to evaluate their affinity with HSA, and it was discovered that Carnosic acid (CA) exhibited the highest affinity while simultaneously revealing multiple binding sites. Further investigation revealed that CA HSA the capability to engineer HSA into spherical and size-tunable nanostructures known as eHSA (Engineering HSA particle), which demonstrated optimized macropinocytosis-dependent cellular internalization. As anticipated, eHSA effectively suppressed the Wnt signaling pathway and reactivated the acquired immune response in vivo. Furthermore, eHSA successfully restored sensitivity to Anti-PD1's anticancer effects in both subcutaneous and orthotopic mouse homograft models of MSI-H CRC, as well as a humanized hu-PBMC patient-derived orthotopic xenograft (PDOX) mouse model of MSI-H CRC, all while maintaining a favorable safety profile. The collective implementation of this clinically viable immune reactivation strategy not only enables the delivery of Wnt inhibitors for CRC therapy, but also serves as an exemplary demonstration of precision-medicine-guided nanopharmaceutical development that effectively harnesses specific cellular indications in pathological states.

Patient tissue-derived FGFR4-variant and wild-type colorectal cancer organoid development and anticancer drug sensitivity testing

ObjectivesFGFR4-variant and wild-type colorectal cancer (CRC) organoids were developed to investigate the effects of FGFR4-targeted drugs, including FGFR4-IN and erdafitinib, on CRC and their possible molecular mechanism. MethodsClinical CRC tissues were collected, seven CRC organoids were developed, and whole exome sequencing (WES) was performed. CRC organoids were cultured and organoid drug sensitivity studies were conducted. Finally, an FGFR4-variant (no wild-type) CRC patient-derived orthotopic xenograft mouse model was developed. Western blot measured ERK/AKT/STAT3 pathway-related protein levels. ResultsWES results revealed the presence of FGFR4-variants in 5 of the 7 CRC organoids. The structural organization and integrity of organoids were significantly altered under the influence of targeted drugs (FGFR4-IN-1 and erdafitinib). The effects of FGFR4 targeted drugs were not selective for FGFR4 genotypes. FGFR4-IN-1 and erdafitinib significantly reduced the growth, diameter, and Adenosine Triphosphate (ATP) activity of organoids. Furthermore, chemotherapeutic drugs, including 5-fluorouracil and cisplatin, inhibited FGFR4-variant and wild-type CRC organoid activity. Moreover, the tumor volume of mice was significantly reduced at week 6, and p-ERK1/2, p-AKT, and p-STAT3 levels were down-regulated following FGFR4-IN-1 and erdafitinib treatment. ConclusionsFGFR4-targeted and chemotherapeutic drugs inhibited the activity of FGFR4-variant and wild-type CRC organoids, and targeted drugs were more effective than chemotherapeutic drugs at the same concentration. Additionally, FGFR4 inhibitors hindered tumorigenesis in FGFR4-variant CRC organoids through ERK1/2, AKT, and STAT3 pathways. However, no wild-type control was tested in this experiment, which need further confirmation in the next study.

Patient-Derived Orthotopic Xenograft Models for High-Grade Pediatric Brain Cancers.

Patient-derived orthotopic xenograft (PDOX) mouse models are considered the gold standard for evidence-based preclinical research in pediatric neuro-oncology. This protocol describes the generation of PDOX models by intracranial implantation of human pediatric brain cancer cells into immune-deficient mice, and their continued propagation to establish cohorts of animals for preclinical research.

DDDR-24. SINGLE-CELL FUNCTIONAL TISSUE PROFILING IDENTIFIES PROMISING CANDIDATES FOR DRUG REPURPOSING AGAINST GLIOBLASTOMA

Abstract There remains an unmet need for novel treatment strategies against glioblastoma that are more effective than the current standard of care. However, the complex tumor microenvironment and genetic heterogeneity of these tumors require novel technologies enabling high-throughput assessment of cellular responses to treatments on a functional level in complex tumor samples with an intact cellular microenvironment. Here, we used a novel single-cell functional tissue profiling platform based on high—throughput microscopy followed by computer vision and machine learning algorithms to screen 70 clinically-approved drugs with good blood-brain-barrier penetration in 35 glioblastoma patient samples that were freshly obtained from surgery. This revealed prognostically relevant subtypes of glioma cell morphologies and promising candidates for drug repurposing. Among the drugs with potent anti-glioma activity, we identified the antidepressant Vortioxetine, that had strong anti-glioma activity across patients independent of the promoter methylation status of O6-methylguanine-DNA methyltransferase (MGMT). Complementrary in vitro studies using patient-derived glioma cell lines and orthotopic glioma xenograft mouse models confirmed the strong anti-glioma activity of Vortioxetine, leading to a notable fraction of long-term surviving mice. Mechanistically, multiplexed transcriptomics and proteomics unveiled that Vortioxetine induced an upregulation of the Activator protein 1 (AP-1) transcription factor family, along with the B-cell translocation gene (BTG) tumor suppressor genes. These findings serve as the cornerstone for future clinical applications of the single-cell functional tissue profiling platform and the potential clinical translation of Vortioxetine as a promising drug-repurposing candidate for glioblastoma patients.

Expanding a precision medicine platform for malignant peripheral nerve sheath tumors: New patient-derived orthotopic xenografts, cell lines and tumor entities.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive soft-tissue sarcomas with a poor survival rate, presenting either sporadically or in the context of neurofibromatosis type 1 (NF1). The histological diagnosis of MPNSTs can be challenging, with different tumors exhibiting great histological and marker expression overlap. This heterogeneity could be partly responsible for the observed disparity in treatment response due to the inherent diversity of the preclinical models used. For several years, our group has been generating a large patient-derived orthotopic xenograft (PDOX) MPNST platform for identifying new precision medicine treatments. Herein, we describe the expansion of this platform using six primary tumors clinically diagnosed as MPNSTs, from which we obtained six additional PDOX mouse models and three cell lines, thus generating three pairs of in vitro-in vivo models. We extensively characterized these tumors and derived preclinical models, including genomic, epigenomic, and histological analyses. Tumors were reclassified after these analyses: three remained as MPNSTs (two being classic MPNSTs), one was a melanoma, another was a neurotrophic tyrosine receptor kinase (NTRK)-rearranged spindle cell neoplasm, and, finally, the last was an unclassifiable tumor bearing neurofibromin-2 (NF2) inactivation, a neuroblastoma RAS viral oncogene homolog (NRAS) oncogenic mutation, and a SWI/SNF-related matrix-associated actin-dependent regulator of chromatin (SMARCA4) heterozygous truncated variant. New cell lines and PDOXs faithfully recapitulated histology, marker expression, and genomic characteristics of the primary tumors. The diversity in tumor identity and their specific associated genomic alterations impacted treatment responses obtained when we used the new cell lines for testing compounds against known altered pathways in MPNSTs. In summary, we present here an extension of our MPNST precision medicine platform, with new PDOXs and cell lines, including tumor entities confounded as MPNSTs in a real clinical scenario. This platform may constitute a useful tool for obtaining correct preclinical information to guide MPNST clinical trials.

High-fat diet promotes tumor growth in the patient-derived orthotopic xenograft (PDOX) mouse model of ER positive endometrial cancer

Endometrial cancer, one of the common gynecological malignancies, is affected by several influencing factors. This study established a unique patient-derived orthotopic xenograft (PDOX) nude mouse model for the study of influencing factors in ER positive endometrial cancer. The aim of this study was to demonstrate that a high-fat diet can affect the growth of ER positive endometrial cancer PDOX model tumors. The tumor tissues were expanded by subcutaneous transplantation in nude mice, and then the subcutaneous tumor tissues were orthotopically implanted into the nude mouse uterus to establish the PDOX model. After modeling, they were divided into high-fat diet group and normal diet group for 8 weeks of feeding, which showed that high-fat diet significantly promoted tumor growth (P < 0.001) and increased the protein expression level of ERα in tumor tissues. This study demonstrates that PDOX models of endometrial cancer can embody the role of dietary influences on tumor growth and that this model has the potential for preclinical studies of cancer promoting factors.

High Clinical Concordance of Drug Resistance in Patient-derived Orthotopic Xenograft (PDOX) Mouse Models: First Step to Validated Precise Individualized Cancer Chemotherapy.

Finding an effective drug for individual cancer patients among the many chemotherapies available and ruling out ineffective drugs are important challenges, especially for patients with advanced cancer. To accomplish this goal, we have pioneered and developed the patient-derived orthotopic xenograft (PDOX) nude mouse model for all cancer types, enabling the discovery and evaluation of novel therapeutics, as well as individualized therapy of patients with cancer. PDOX models can more precisely reproduce the original tumor microenvironment (TME) compared to subcutaneous-implanted xenografts including patient-derived xenograft (PDX) models. The present review presents the concordance of drug resistance in individual cancer patients and their PDOX models. There are 28 PDOX publications with 12 PDOX models from patients who were treated with chemotherapy. Sixteen chemotherapeutics were administrated to these patients and all of them were clinically ineffective. In PDOX models established from these patients' tumors, fourteen chemotherapeutics were resistant with a concordance rate of 88%. PDOX models should be established as early as possible from patients to predict and improve outcome. PDOX models mimic the clinical tumor aggressiveness, therefore enabling a high concordance with clinical outcomes. The present review shows a high concordance for drug resistance between cancer patients and their corresponding PDOX models. Future studies will include prospective clinical trials comparing both drug efficacy and resistance in patients and their PDOX models.

P10.22.B COMBINATION OF ARTS MIMETICS WITH AUTOPHAGY INHIBITORS AS A NOVEL THERAPEUTIC STRATEGY TO OVERCOME APOPTOSIS RESISTANCE IN GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GBM) is the most common malignant brain tumour in adults. The current standard of care for GBM patients consists of maximal surgical resection followed by chemotherapy and radiation. Despite such an aggressive treatment approach, the average 5-year survival rate for GBM patients is approximately 5%. One of the main reasons for poor therapy response in the GBM is resistance to programmed cell death, otherwise known as apoptosis. One of the mechanisms behind apoptosis resistance relies on the upregulation of inhibitor of apoptosis proteins (IAPs) which are known to hinder pro-apoptotic proteins and allow cell survival under abnormal conditions such as severe DNA damage. However, there are natural IAP antagonists such as ARTS which are capable of stimulating cell death by promoting the degradation of IAPs. To harness the pro-apoptotic function of IAP antagonists, small peptide mimetics of ARTS have been developed. This project investigates the efficacy of utilizing ARTS mimetics as a novel strategy to overcome apoptosis resistance in the GBM treatment. MATERIAL AND METHODS This study utilizes patient-derived cells with exogenous gain-of-function (GOF) overexpression to introduce full-length ARTS protein as well as pharmacological small peptide mimetics of ARTS to evaluate the response of GBM cells following the promotion of ARTS activity. We also employ a patient-derived orthotopic xenograft mouse model to test novel combinatorial strategies in vivo, where we used a nanoparticle drug delivery system to ensure treatment delivery across the blood-brain barrier (BBB). RESULTS We found that patient-derived GBM cells were highly resistant to ARTS-induced apoptosis. Further molecular investigation unveiled a novel role of ARTS in the upregulation of autophagy as a resistance mechanism. A combination of ARTS GOF or mimetic peptides with autophagy inhibitors mitigated resistance to ARTS-mediated apoptosis and drastically decreased GBM cell proliferation. This combination was further tested in the patient-derived xenograft mouse model, which demonstrated the successful delivery of ARTS mimetic peptides and autophagy inhibitors through the BBB via nanoparticle lipid carriers. Animals treated with a combination of the autophagy inhibitor Spautin-1 and ARTS mimetic peptides demonstrated significantly prolonged survival, confirming the therapeutic potential of such combinatorial approach for GBM treatment. CONCLUSION Altogether, these findings uncover a previously unknown role of ARTS in autophagy regulation and provide evidence that the combination of IAP-antagonist mimetics with autophagy inhibitors can be an effective strategy against highly aggressive GBM brain tumours. This work was supported by the Health Sciences Centre Foundation (HSCF).

Relationship between efficacy of phase I/II drug ispinesib in medulloblastoma, molecular subtypes, and p53-mutant status.

e14001 Background: KIF11, a mitotic kinesin, is responsible for assembly and maintenance of mitotic spindle during mitosis. Tumor cells can upregulate KIF11 to enhance tumor proliferation. Many cancers overexpress KIF11, including medulloblastoma (MB), the most common malignant brain tumor in children. MB is molecularly classified into 4 molecular subtypes: sonic-hedgehog (SHH)-activated MB, Wingless (WNT)-activated, and less characterized Group 3 and 4 subtypes. Group 3 and 4 MBs are the most aggressive subtypes and Group 3 MBs have the worst survival outcome. p53 mutation defines a high-risk group in MB patients. We evaluated the efficacy of a Phase I/II KIF11 inhibitor, ispinesib on MB. Methods: We employed in-vitro proliferation assays (CCK), flow cytometry (Annexin V, Propidium Iodide), western blot, Hoechst staining, and two patient-derived orthotopic xenograft (PDOX) mouse models for in-vivo drug testing. Results: Ispinesib effectively inhibited proliferation at nanomolar concentrations for all 5 MB cell lines. A comparison of IC50 (day 4) indicated that p53-mutational-status did not influence sensitivity to ispinesib. In all five cell lines, regardless of p53 mutational status, ispinesib led to G2/M arrest. However, different p53-mutational-status led to different modes of cell death. High levels of necrotic cell death were observed in p53-mutant, while apoptosis was the predominant mode of cell death in p53-wildtype, on flow cytometry. Apoptotic nuclei were more significantly observed in p53-wildtype (p = 0.002) while p53-mutant cell line demonstrated more micronuclei (p = 0.0302) and multi-nucleation (p= 0.0161), compared to respective DMSO-control cells on Hoechst staining. Ispinesib has demonstrated effective blood brain barrier penetration in brain xenografts. We compared 2 PDOX models of MB. Ispinesib significantly improved survival outcome of Group 3 MB (Control=6, Treated=7, Log-rank p=0.0003), but worsened the survival outcome of p53-mutant SHH MB (Control=14, Treated=11, Log-rank p=0.169). This differential efficacy of ispinesib was recapitulated in the matched 3D-neurosphere cultures of both PDOX models. Cancer-stem-cell (CSC)-enriched neurospheres derived from aggressive Group 3 subtype demonstrated susceptibility to 7-day in-vitro treatment with ispinesib (AUC=0.7743). Monolayer tumor cells demonstrated similar susceptibility (AUC=0.7757). Area under curve (AUC)&gt;0.7 was the significant cut-off. In contrast, both CSC-enriched neurospheres (AUC=0.379) and monolayer tumor cells (AUC=0.491) derived from p53-mutant SHH MB did not demonstrate susceptibility to ispinesib, consistent with matching PDOX models. Conclusions: Preclinically, our results indicate feasibility of ispinesib treatment in MB, specifically aggressive Group 3 MB. Importantly, p53-mutant SHH MB represents a poor molecular-subtype candidate for ispinesib therapy.

Highly Selective Targeting of Pancreatic Cancer in the Liver with a Near-Infrared Anti-MUC5AC Probe in a PDOX Mouse Model: A Proof-of-Concept Study.

Accurately identifying metastatic disease is critical to directing the appropriate treatment in pancreatic cancer. Mucin 5AC is overexpressed in pancreatic cancer but absent in normal pancreas tissue. The present proof-of-concept study demonstrates the efficacy of an anti-mucin 5AC antibody conjugated to an IR800 dye (MUC5AC-IR800) to preferentially label a liver metastasis of pancreatic cancer (Panc Met) in a unique patient-derived orthotopic xenograft (PDOX) model. In orthotopic models, the mean tumor to background ratio was 1.787 (SD ± 0.336), and immunohistochemistry confirmed the expression of MUC5AC within tumor cells. MUC5AC-IR800 provides distinct visualization of pancreatic cancer liver metastasis in a PDOX mouse model, demonstrating its potential utility in staging laparoscopy and fluorescence-guided surgery.

In vivo loss of tumorigenicity in a patient-derived orthotopic xenograft mouse model of ependymoma.

Ependymomas (EPN) are the third most common malignant brain cancer in children. Treatment strategies for pediatric EPN have remained unchanged over recent decades, with 10-year survival rates stagnating at just 67% for children aged 0-14 years. Moreover, a proportion of patients who survive treatment often suffer long-term neurological side effects as a result of therapy. It is evident that there is a need for safer, more effective treatments for pediatric EPN patients. There are ten distinct subgroups of EPN, each with their own molecular and prognostic features. To identify and facilitate the testing of new treatments for EPN, in vivo laboratory models representative of the diverse molecular subtypes are required. Here, we describe the establishment of a patient-derived orthotopic xenograft (PDOX) model of posterior fossa A (PFA) EPN, derived from a metastatic cranial lesion. Patient and PDOX tumors were analyzed using immunohistochemistry, DNA methylation profiling, whole genome sequencing (WGS) and RNA sequencing. Both patient and PDOX tumors classified as PFA EPN by methylation profiling, and shared similar histological features consistent with this molecular subgroup. RNA sequencing revealed that gene expression patterns were maintained across the primary and metastatic tumors, as well as the PDOX. Copy number profiling revealed gains of chromosomes 7, 8 and 19, and loss of chromosomes 2q and 6q in the PDOX and matched patient tumor. No clinically significant single nucleotide variants were identified, consistent with the low mutation rates observed in PFA EPN. Overexpression of EZHIP RNA and protein, a common feature of PFA EPN, was also observed. Despite the aggressive nature of the tumor in the patient, this PDOX was unable to be maintained past two passages in vivo. Others who have successfully developed PDOX models report some of the lowest success rates for EPN compared to other pediatric brain cancer types attempted, with loss of tumorigenicity not uncommon, highlighting the challenges of propagating these tumors in the laboratory. Here, we discuss our collective experiences with PFA EPN PDOX model generation and propose potential approaches to improve future success in establishing preclinical EPN models.

Synergistic effects of concurrent photodynamic therapy with indocyanine green and chemotherapy in hepatocellular carcinoma cell lines and mouse models.

Photodynamic therapy (PDT) using an 808nm laser irradiation with indocyanine green (ICG) has shown tumoricidal effects in a hepatocellular (HCC) orthotopic xenograft model. Recently, combining PDT with concurrent chemotherapy has shown synergistic outcomes and a better therapeutic effect for cancer treatment. In the present study, we utilized a combination of chemotherapy drugs and PDT using ICG in vitro and in vivo in a patient-derived orthotopic xenograft (PDoX) model. We independently performed PDT and chemotherapy with sorafenib or doxorubicin in the Huh-7 and Hep3b cell lines by increasing the sorafenib or doxorubicin concentration and increasing the total energy of 808nm light. Subsequently, we combined the two treatments to confirm the effects on cell viability. The combination index (CI) was evaluated in vitro, and thereafter, in the HCC PDoX mouse model, 808nm laser irradiation with intravenously injected ICG and chemotherapy using doxorubicin were performed for twelve days. The viability of the Huh-7 and Hep3B cell lines decreased rapidly as the concentration of sorafenib or doxorubicin increased and as the total energy of 808nm light increased. The cell viability of the Huh-7 and Hep3b cell lines with combined PDT and chemotherapy was less than that with PDT or chemotherapy alone. The CI was <1 in the sorafenib- or doxorubicin-treated Huh-7 and Hep3b cell lines. In the HCC PDoX mouse model, tumor size was markedly decreased, and complete remission achieved compared to that of the single chemotherapy or PDT and control groups. The synergistic effect of concurrent PDT and chemotherapy in the HCC cell line and PDoX model was confirmed with no definite adverse effect. Concurrent PDT and chemotherapy could be applied in further preclinical studies.

Targeting Autophagy With the Synergistic Combination of Chloroquine and Rapamycin as a Novel Effective Treatment for Well-differentiated Liposarcoma.

Liposarcoma is a type of soft-tissue sarcoma arising from fat tissue. It is relatively common among soft-tissue sarcomas. Chloroquine (CQ), an antimalarial drug, can inhibit autophagy and induce apoptosis in cancer cells. Rapamycin (RAPA) is an inhibitor of mTOR. The combination of RAPA and CQ is a strong inhibitor of autophagy. Previously, we showed that the combination of RAPA and CQ was effective against a de-differentiated liposarcoma patient-derived orthotopic xenograft (PDOX) mouse model. In the present study, we investigated the mechanism of efficacy of the combination of RAPA and CQ to target autophagy in a well-differentiated liposarcoma (WDLS) cell line in vitro. The human WDLS cell line 93T449 was used. The WST-8 assay was used to test the cytotoxicity of RAPA and CQ. Western blotting was used to detect microtubule-associated protein light chain 3-II (LC3-II) which is a component of autophagosomes. Immunostaining of LC3-II was also performed for autophagosome analysis. Τhe TUNEL assay was used to detect apoptotic cells, and apoptosis-positive cells were counted in three randomly selected microscopic fields for statistical validation. RAPA alone and CQ alone inhibited the viability of 93T449 cells. The combination of RAPA and CQ inhibited 93T449 cell viability significantly more than either drug alone and increased the number of autophagosomes which led to extensive apoptosis. The combination of RAPA and CQ increased the number of autophagosomes which led to apoptosis in 93T449 WDLS cells, suggesting novel effective treatment for this recalcitrant cancer by targeting autophagy.

Oral-recombinant Methioninase in Combination With Rapamycin Eradicates Osteosarcoma of the Breast in a Patient-derived Orthotopic Xenograft Mouse Model

Primary osteosarcoma of the breast is a very rare malignancy that shares histological features with osteosarcoma. It is also highly sensitive to methionine restriction due to methionine addiction. We previously established a patient-derived orthotopic xenograft (PDOX) nude-mouse model derived from tumor tissue of a patient with primary mammary osteosarcoma. In the present study, we investigated the efficacy of oral-recombinant methioninase (o-rMETase), combined with rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) kinase, on a mammary osteosarcoma PDOX nude-mouse model. The PDOX mouse model was established by surgically transplanting a specimen of primary osteosarcoma of the breast into the mammary gland of nude mice. Mice implanted with tumors were randomly divided into four groups: Control group, N=5; rapamycin-treated group, N=5; o-rMETase-treated group, N=5; and a group treated with the combination of o-rMETase and rapamycin, N=5. Mice were treated for 2 weeks after transplantation, and tumor volume was measured during the treatment period. Treatment with the combination of rapamycin and o-rMETase eradicated the osteosarcoma of the breast compared to the untreated control (p=0.000008). o-rMETase alone did not significantly inhibit tumor growth, and rapamycin alone only partially inhibited the tumor (p=0.78 and p=0.018, respectively) compared to the untreated control. There was not a significant difference in mouse weight between the groups. The combination of rapamycin and o-rMETase was highly effective against primary osteosarcoma of the breast in a PDOX model, suggesting a future clinical strategy for this rare cancer type that currently has no first-line treatment.

Review: Precise sarcoma patient-derived orthotopic xenograft (PDOX) mouse models enable identification of novel effective combination therapies with the cyclin-dependent kinase inhibitor palbociclib: A strategy for clinical application.

IntroductionSarcomas are rare heterogeneous malignant tumors that originate and develop in soft tissue or bone. Effective treatment for sarcomas is still limited to traditional chemotherapy and surgery that are often ineffective for recurrent disease. Cyclin-dependent kinases (CDKs) promote abnormal cell cycling and cell division in many cancers including sarcomas. Therefore, our hypothesis was that CDK inhibitors may be useful candidates for sarcoma treatment. Patient-derived orthotopic xenograft (PDOX) mouse models mimic the clinical disease for all major cancer types and have identified effective treatments that hold much clinical promise. The present report reviews sarcoma PDOX models that we have established for their potential to discover effective combination treatments based on CDK inhibitors for recalcitrant sarcoma.MethodsWe have previously reported six sarcoma PDOX studies evaluating the CDK inhibitor palbociclib on sarcoma, including osteosarcoma, Ewing sarcoma, de-differentiated liposarcoma, and peritoneal metastatic leiomyosarcoma.ResultsPalbociclib monotherapy significantly inhibited, but not regressed, the PDOX growth of osteosarcoma, Ewing sarcoma, de-differentiated liposarcoma, and peritoneal metastatic leiomyosarcoma. A combination of palbociclib and a mammalian target of rapamycin (mTOR) inhibitor, everolimus, significantly inhibited, but did not regress, the PDOX growth of osteosarcoma. Combinations of palbociclib with a multikinase inhibitor, sorafenib, and palbociclib combined with recombinant methioninase were effective and regressed the osteosarcoma and de-differentiated liposarcoma PDOX models, respectively.ConclusionsNovel effective drug combinations using the CDK inhibitor palbociclib were identified in PDOX models of the major types of sarcomas. Methionine restriction effected by methioninase increased the efficacy of palbociclib. Combination therapy with palbociclib is a promising future strategy for improved sarcoma therapy in the clinic.

Abstract 5466: Efficacy of ASC60, an oral fatty acid synthase inhibitor, in two tumor mouse models

Abstract Background: Fatty acid synthase (FASN) is a key enzyme controlling endogenous lipid biosynthesis and overexpressed in many cancers. Studies have shown that FASN inhibition can disrupt lipid synthesis in tumor cells and disrupt tumor-associated signal transduction, suggesting FASN inhibition can serve as a potential treatment for cancer. Here we report the in vivo efficacy data of ASC60, a small molecular FASN inhibitor, either as a single agent or in combination with mouse programmed cell death-1 (mPD-1) antibody in two tumor mouse models. Methods: In the MC38 mouse model, mice were inoculated subcutaneously with MC38 cells. When the average tumor volume reached approximately 62 mm3, mice were treated with ASC60 (60, and 100 mg/kg) or vehicle once a day (QD) for 3 weeks in combination mPD-1 antibody twice a week (1 mg/kg, BIW) for 2 weeks. In patient derived orthotopic xenograft (PDOX) brain metastasis mouse model, luciferase labelled breast cancer cells (LD1-2009-362541-Luc) were transplanted into the brain of nude mice. Tumor volumes were estimated by the luciferase reporter signal. When the average signal reached 4.12X107 photon/sec, mice were treated with ASC60 (20, 60, and 100 mg/kg) or vehicle QD for 4 weeks. In both mouse models, body weights and tumor volumes were measured regularly, and tumor growth inhibitions (TGI) of different groups were compared. Results: All animals showed a gradually increase in body weight during the study and no significant difference of body weight or body weight change was found among different groups. In the MC38 colon cancer mouse model, mice treated with ASC60 (60 or 100 mg/kg) in combination with mPD-1 antibody showed higher TGI values compared to those treated with mPD-1 alone (91.86%, 99.88% vs 89.84%) on Day 13, indicating combination with ASC60 would enhance the antitumor activity of mPD-1 antibody. In PDOX mouse model, mice treated with ASC60 (20, 60, and 100 mg/kg) showed dose dependent TGI values of 10.83%, 36.95% and 49.21% on Day 13 and of 6.46%, 21.27% and 30.47% on Day 27, respectively. ASC60 also showed a significant improvement in life span (51.5 days in ASC60 60 mg/kg vs 39 days in vehicle control). Conclusion: Previous in vitro testing showed that inhibition of palmitate synthesis by ASC60 was 137-fold lower in mouse than in human (IC50: 2.05 µM in mouse vs 0.015 µM in human). However, results of the present studies showed that ASC60 could suppress tumor growth and/or expand lifespan in the two tumor mouse models either alone or in combination with mPD-1 antibody. ASC60 will possibly demonstrate better efficacies in human. Citation Format: Jinzi J. Wu, Handan He. Efficacy of ASC60, an oral fatty acid synthase inhibitor, in two tumor mouse 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 5466.

MODL-29. Molecular Landscape of a comprehensive panel of pediatric brain cancer Patient-derived orthotopic xenograft (PDOX) models inform unique targets for drug responsiveness

Abstract Brain tumor is a leading cause of cancer related death in children. In addition to replicating histopathology, animal models faithfully replicating genetic/epigenetic abnormalities, molecular subtypes and broad inter-tumoral heterogeneities are needed. Through direct implantation of patient surgical or autopsied tumor tissues into matching locations in the brains of SCID mice, we developed a panel of 150 PDOX mouse models. Here, we report the analysis of 74 of the 150 PDOX models, 45 matching patient tissues and 60 non-tumorigenic samples to a well-annotated reference cohort of 2,801 methylation profiles of primary brain tumors. Our data showed that the lack of tumorigenicity was neither correlated with molecular subtypes nor predicted by low cell viabilities of the patient samples. Methylation profiling identified PDOX models representing nearly a full spectrum of molecular subtypes of pediatric brain tumors including GBM, medulloblastoma, ependymoma and ATRT. Direct comparison with the original patient tumors confirmed the replication of molecular subtypes. ONCOplot [FB1] analysis of PDOX models derived from matching pairs of primary and recurrent tumors (n=8) revealed close clustering with the patient tumors. Investigation of metastatic properties was performed in 13 MB models by harvesting and sub-transplanting matching PDOX primary tumors in the cerebella and metastatic tumors in the spinal cords. To confirm the potential and power of PDOX models in preclinical drug testing, we applied fractionated radiation (2 Gy/day x 5 days) and optimized multi-agent combinatory chemotherapies in MB models of the four major subgroups. High-throughput combination drug screening with ~ 8,000 drugs in PDOX-derived GBM cell lines and primary cultures of MB PDOX cells identified a library of ~ 3,500 drugs that were active in pediatric brain tumors. In summary, this study provides detailed information on molecular subclassification of a uniquely large cohort of PDOX models to serve as essential tools for brain tumor research.

Anti-mucin 4 fluorescent antibody brightly targets colon cancer in patient-derived orthotopic xenograft mouse models: A proof-of-concept study for future clinical applications

BackgroundThere is a high rate of positive surgical margins with resection of liver metastases in colorectal cancer (CRC). The present study reports using a fluorescent anti-mucin 4 (MUC4) antibodies to label primary CRC and liver metastases to better visualize tumor margins in mouse models. MethodsWestern blotting for MUC4 protein expression of normal colon and CRC tumor lysates was performed. Orthotopic primary and liver metastatic CRC mouse models received anti-MUC4 antibody conjugated to IR800 (MUC4-IR800). Mice were sacrificed and imaged after 48 hours. ResultsWestern blotting demonstrated increased MUC4 expression in a human CRC cell line and patient-derived primary and liver-metastatic CRCs. The LS174T orthotopic primary CRC model tumor to background ratio (TBR) was 2.04 (±0.35). The patient-derived orthotopic xenograft (PDOX) primary CRC model TBR was 2.17 (±0.35). The PDOX liver metastasis model TBR was 1.56 (±0.53). ConclusionMUC4-IR800 provided bright labeling of primary and liver tumors in CRC orthotopic mouse models, demonstrating their future clinical potential for margin visualization in fluorescence guided surgery.

1,3,5-triazines inhibit osteosarcoma and avert lung metastasis in a patient-derived orthotopic xenograft mouse model with favorable pharmacokinetics.

Objective(s): Osteosarcoma is a major solid malignant tumor of bone, possessing significant burden on healthcare due to non-availability of specific anticancer agents. The current study was conducted to identify novel 1,3,5-triazine derivatives against osteosarcoma. Materials and Methods:The compounds were synthesized in a straight-forward two-step reaction and subsequently tested against PI3K and mTOR kinase and anticancer activity against osteosarcoma cells (MG-63, U2-OS, and Saos-2). The effect of the most potent compound was evaluated on apoptosis and cell phase of Saos-2 cells. The pharmacological activity was further established in the patient-derived orthotopic xenograft (PDOX) mouse model. Results:The developed compounds 8 (a-f) showed significant inhibitory activities against PI3K, mTOR, and OS cells. Among the tested series, compound 8a showed highly potent PI3K/mTOR inhibitory activity with significant anticancer activity against Saos-2 cells compared with Imatinib as standard. It also induces apoptosis and causes G2/M arrest in Saos-2 cells. Compound 8a significantly improved body weight, reduced tumor volume, and inhibited lung metastasis in athymic nude mice in a PDOX mouse model. It also showed optimal pharmacokinetic parameters in SD rats. Conclusion:In summary, 1,3,5-triazine analogs were identified as new PI3K/mTOR inhibitors against osteosarcoma.

Oral-recombinant Methioninase Lowers the Effective Dose and Eliminates Toxicity of Cisplatinum for Primary Osteosarcoma of the Mammary Gland in a Patient-derived Orthotopic Xenograft Mouse Model.

Primary osteosarcoma of the mammary gland is a very rare disease, accounting for under 1% of all mammary gland malignancies. There is no established first-line treatment, and prognosis is poor compared to conventional breast cancer. We previously demonstrated the efficacy of cisplatinum and eribulin in a patient-derived orthotopic xenograft (PDOX) mouse model of primary breast osteosarcoma. However, these drugs show significant clinical toxicity. All cancers are addicted to methionine (Hoffman effect). In the present study, we determined whether methionine restriction with oral recombinant methioninase (o-rMETase) would lower the effective dose of cisplatinum in a PDOX model of primary osteosarcoma of the mammary gland, thereby reducing its toxicity. Mouse PDOX models of primary osteosarcoma of the breast were randomized into the following groups: control; cisplatinum (weekly at 3 or 6 mg/kg); twice-daily o-rMETase; or o-rMETase combined with 3 mg/kg cisplatinum, with treatment for 2 weeks. Cisplatinum at 6 mg/kg significantly inhibited breast osteosarcoma growth compared with the untreated control and mice treated with 3 mg/kg cisplatinum (p=0.01 and 0.009, respectively). There was no significant difference in tumor growth between mice treated with cisplatinum at 3 mg/kg and the control (p=0.16). Combination therapy with cisplatinum at 3 mg/kg and twice daily o-rMETase regressed the osteosarcoma of the mammary gland (p=0.009), similar to the inhibition by cisplatinum at 6 mg/kg alone. Cisplatinum at 6 mg/kg caused a significant loss of mouse body weight, compared to the control (p=0.02). There was no significant body-weight loss with the combination therapy of o-rMETase and cisplatinum at 3 mg/kg, compared to the untreated control. o-rMETase halved the effective dose of cisplatinum, thereby eliminating cisplatinum toxicity, demonstrating a future clinical strategy for therapy of osteosarcoma of the breast.