Tumor Evolution and Drug Response in Patient-Derived Organoid Models of Bladder Cancer.

Bladder cancer is the fifth most prevalent cancer in the U.S., yet is understudied, and few laboratory models exist that reflect the biology of the human disease. Here, we describe a biobank of patient-derived organoid lines that recapitulates the histopathological and molecular diversity of human bladder cancer. Organoid lines can be established efficiently from patient biopsies acquired before and after disease recurrence and are interconvertible with orthotopic xenografts. Notably, organoid lines often retain parental tumor heterogeneity and exhibit a spectrum of genomic changes that are consistent with tumor evolution in culture. Analyses of drug response using bladder tumor organoids show partial correlations with mutational profiles, as well as changes associated with treatment resistance, and specific responses can be validated using xenografts in vivo. Our studies indicate that patient-derived bladder tumor organoids represent a faithful model system for studying tumor evolution and treatment response in the context of precision cancer medicine.

Synergic activity of FGFR2 and MEK inhibitors in the treatment of FGFR2-amplified cancers of unknown primary.

Patients with cancer of unknown primary (CUP) carry the double burden of an aggressive disease and reduced access to therapies. Experimental models are pivotal for CUP biology investigation and drug testing. We derived two CUP cell lines (CUP#55 and #96) and corresponding patient-derived xenografts (PDXs), from ascites tumor cells. CUP cell lines and PDXs underwent histological, immune-phenotypical, molecular, and genomic characterization confirming the features of the original tumor. The tissue-of-origin prediction was obtained from the tumor microRNA expression profile and confirmed by single-cell transcriptomics. Genomic testing and fluorescence in situ hybridization analysis identified FGFR2 gene amplification in both models, in the form of homogeneously staining region (HSR) in CUP#55 and double minutes in CUP#96. FGFR2 was recognized as the main oncogenic driver and therapeutic target. FGFR2-targeting drug BGJ398 (infigratinib) in combination with the MEK inhibitor trametinib proved to be synergic and exceptionally active, both in vitro and in vivo. The effects of the combined treatment by single-cell gene expression analysis revealed a remarkable plasticity of tumor cells and the greater sensitivity of cells with epithelial phenotype. This study brings personalized therapy closer to CUP patients and provides the rationale for FGFR2 and MEK targeting in metastatic tumors with FGFR2 pathway activation.

Single-cell RNA sequencing reveals anti-tumor potency of CD56+ NK cells and CD8+ T cells in humanized mice via PD-1 and TIGIT co-targeting.

In solid tumors, the exhaustion of natural killer (NK) cells and cytotoxic T cells in the immunosuppressive tumor microenvironment poses challenges for effective tumor control. Conventional humanized mouse models of hepatocellular carcinoma patient-derived xenografts (HCC-PDX) encounter limitations in NK cell infiltration, hindering studies on NK cell immunobiology. Here, we introduce an improved humanized mouse model with restored NK cell reconstitution and infiltration in HCC-PDX, coupled with single-cell RNA sequencing (scRNA-seq) to identify potential anti-HCC treatments. A single administration of adeno-associated virus carrying human interleukin-15 reinstated persistent NK cell reconstitution and infiltration in HCC-PDX in humanized mice. scRNA-seq revealed NK cell and T cell subpopulations with heightened PDCD1 and TIGIT levels. Notably, combination therapy with anti-PD-1 and anti-TIGIT antibodies alleviated HCC burden in humanized mice, demonstrating NK cell-dependent efficacy. Bulk-RNA sequencing analysis also revealed significant alterations in the tumor transcriptome that may contribute to further resistance after combination therapy, warranting further investigations. As an emerging strategy, ongoing clinical trials with anti-PD-1 and anti-TIGIT antibodies provide limited data. The improved humanized mouse HCC-PDX model not only sheds light on the pivotal role of NK cells but also serves as a robust platform for evaluating safety and anti-tumor efficacy of combination therapies and other potential regimens, complementing clinical insights.

Proteins in Tumor-Derived Plasma Extracellular Vesicles Indicate Tumor Origin.

Cancer-derived extracellular vesicles (EVs) promote tumorigenesis, premetastatic niche formation, and metastasis via their protein cargo. However, the proteins packaged by patient tumors into EVs cannot be determined in vivo because of the presence of EVs derived from other tissues. We therefore developed a cross-species proteomic method to quantify the human tumor-derived proteome of plasma EVs produced by patient-derived xenografts of four cancer types. Proteomic profiling revealed individualized packaging of novel protein cargo, and machine learning accurately classified the type of the underlying tumor.

Stable isotope-resolved metabolomics analyses of metabolic phenotypes reveal variable glutamine metabolism in different patient-derived models of non-small cell lung cancer from a single patient.

INTRODUCTION: Stable isotope tracers have been increasingly used in preclinical cancer model systems, including cell culture and mouse xenografts, to probe the altered metabolism of a variety of cancers, such as accelerated glycolysis and glutaminolysis and generation of oncometabolites. Comparatively little has been reported on the fidelity of the different preclinical model systems in recapitulating the aberrant metabolism of tumors. OBJECTIVES: We have been developing several different experimental model systems for systems biochemistry analyses of non-small cell lung cancer (NSCLC1) using patient-derived tissues to evaluate appropriate models for metabolic and phenotypic analyses. METHODS: To address the issue of fidelity, we have carried out a detailed Stable Isotope-Resolved Metabolomics study of freshly resected tissue slices, mouse patient derived xenografts (PDXs), and cells derived from a single patient using both 13C6-glucose and 13C5,15N2-glutamine tracers. RESULTS: Although we found similar glucose metabolism in the three models, glutamine utilization was markedly higher in the isolated cell culture and in cell culture-derived xenografts compared with the primary cancer tissue or direct tissue xenografts (PDX). CONCLUSIONS: This suggests that caution is needed in interpreting cancer biochemistry using patient-derived cancer cells in vitro or in xenografts, even at very early passage, and that direct analysis of patient derived tissue slices provides the optimal model for ex vivo metabolomics. Further research is needed to determine the generality of these observations.

Species-Deconvolved Proteomics for In Situ Investigation of Tumor-Stroma Interactions after Treatment of Pancreatic Cancer Patient-Derived Xenografts with Combined Gemcitabine and Paclitaxel.

Tumor-stroma interactions are critical in pancreatic ductal adenocarcinoma (PDAC) progression and therapeutics. Patient-derived xenograft (PDX) models recapitulate tumor-stroma interactions, but the conventional antibody-based immunoassay is inadequate to discriminate tumor and stromal proteins. Here, we describe a species-deconvolved proteomics approach embedded in IonStar that can unambiguously quantify the tumor (human-derived) and stromal (mouse-derived) proteins in PDX samples, enabling unbiased investigation of tumor and stromal proteomes with excellent quantitative reproducibility. With this strategy, we studied tumor-stroma interactions in PDAC PDXs that responded differently to Gemcitabine combined with nab-Paclitaxel (GEM+PTX) treatment. By analyzing 48 PDX animals 24 h/192 h after treatment with/without GEM+PTX, we quantified 7262 species-specific proteins under stringent cutoff criteria, with high reproducibility. For the PDX sensitive to GEM+PTX, the drug-dysregulated proteins in tumor cells were involved in suppressed oxidative phosphorylation and the TCA cycle, and in the stroma, inhibition of glycolytic activity was predominant, suggesting a relieved reverse Warburg effect by the treatment. In GEM+PTX-resistant PDXs, protein changes suggested extracellular matrix deposition and activation of tumor cell proliferation. Key findings were validated by immunohistochemistry (IHC). Overall, this approach provides a species-deconvolved proteomic platform that could advance cancer therapeutic studies by enabling unbiased exploration of tumor-stroma interactions in the large number of PDX samples required for such investigations.

Antitumor activity of the PI3K δ-sparing inhibitor MEN1611 in PIK3CA mutated, trastuzumab-resistant HER2 + breast cancer.

PURPOSE: Dysregulation of the PI3K pathway is one of the most common events in breast cancer. Here we investigate the activity of the PI3K inhibitor MEN1611 at both molecular and phenotypic levels by dissecting and comparing its profile and efficacy in HER2 + breast cancer models with other PI3K inhibitors. METHODS: Models with different genetic backgrounds were used to investigate the pharmacological profile of MEN1611 against other PI3K inhibitors. In vitro studies evaluated cell viability, PI3K signaling, and cell death upon treatment with MEN1611. In vivo efficacy of the compound was investigated in cell line- and patient-derived xenografts models. RESULTS: Consistent with its biochemical selectivity, MEN1611 demonstrated lower cytotoxic activity in a p110δ-driven cellular model when compared to taselisib, and higher cytotoxic activity in the p110ß-driven cellular model when compared to alpelisib. Moreover, MEN1611 selectively decreased the p110α protein levels in PIK3CA mutated breast cancer cells in a concentration- and proteasome-dependent manner. In vivo, MEN1611 monotherapy showed significant and durable antitumor activity in several trastuzumab-resistant PIK3CA-mutant HER2 + PDX models. The combination of trastuzumab and MEN1611 significantly improved the efficacy compared to single agent treatment. CONCLUSIONS: The profile of MEN1611 and its antitumoral activity suggest an improved profile as compared to pan-inhibitors, which are limited by a less than ideal safety profile, and isoform selective molecules, which may potentially promote development of resistance mechanisms. The compelling antitumor activity in combination with trastuzumab in HER2 + trastuzumab-resistant, PIK3CA mutated breast cancer models is at the basis of the ongoing B-Precise clinical trial (NCT03767335).

In vivo activity of the dual SYK/FLT3 inhibitor TAK-659 against pediatric acute lymphoblastic leukemia xenografts.

BACKGROUND: While children with acute lymphoblastic leukemia (ALL) experience close to a 90% likelihood of cure, the outcome for certain high-risk pediatric ALL subtypes remains dismal. Spleen tyrosine kinase (SYK) is a prominent cytosolic nonreceptor tyrosine kinase in pediatric B-lineage ALL (B-ALL). Activating mutations or overexpression of Fms-related receptor tyrosine kinase 3 (FLT3) are associated with poor outcome in hematological malignancies. TAK-659 (mivavotinib) is a dual SYK/FLT3 reversible inhibitor, which has been clinically evaluated in several other hematological malignancies. Here, we investigate the in vivo efficacy of TAK-659 against pediatric ALL patient-derived xenografts (PDXs). METHODS: SYK and FLT3 mRNA expression was quantified by RNA-seq. PDX engraftment and drug responses in NSG mice were evaluated by enumerating the proportion of human CD45+ cells (%huCD45+ ) in the peripheral blood. TAK-659 was administered per oral at 60 mg/kg daily for 21 days. Events were defined as %huCD45+ ≥ 25%. In addition, mice were humanely killed to assess leukemia infiltration in the spleen and bone marrow (BM). Drug efficacy was assessed by event-free survival and stringent objective response measures. RESULTS: FLT3 and SYK mRNA expression was significantly higher in B-lineage compared with T-lineage PDXs. TAK-659 was well tolerated and significantly prolonged the time to event in six out of eight PDXs tested. However, only one PDX achieved an objective response. The minimum mean %huCD45+ was significantly reduced in five out of eight PDXs in TAK-659-treated mice compared with vehicle controls. CONCLUSIONS: TAK-659 exhibited low to moderate single-agent in vivo activity against pediatric ALL PDXs representative of diverse subtypes.

In vivo activity of the dual PI3Kδ and PI3Kγ inhibitor duvelisib against pediatric acute lymphoblastic leukemia xenografts.

BACKGROUND: Acute lymphoblastic leukemia (ALL) remains one of the most common causes of cancer-related mortality in children. Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases, and aberrations in the PI3K pathway are associated with several hematological malignancies, including ALL. Duvelisib (Copiktra) is an orally available, small molecule dual inhibitor of PI3Kδ and PI3Kγ, that is Food and Drug Administration (FDA) approved for the treatment of relapsed/refractory chronic lymphocytic leukemia and small lymphocytic lymphoma. Here, we report the efficacy of duvelisib against a panel of pediatric ALL patient-derived xenografts (PDXs). PROCEDURES: Thirty PDXs were selected for a single mouse trial based on PI3Kδ (PIK3CD) and PI3Kγ (PIK3CG) expression and mutational status. PDXs were grown orthotopically in NSG (NOD.Cg-Prkdcscid IL2rgtm1Wjl /SzJAusb) mice, and engraftment was evaluated by enumerating the proportion of human versus mouse CD45+ cells (%huCD45+ ) in the peripheral blood. Treatment commenced when the %huCD45+ reached greater than or equal to 1%, and events were predefined as %huCD45+ greater than or equal to 25% or leukemia-related morbidity. Duvelisib was administered per oral (50 mg/kg, twice daily for 28 days). Drug efficacy was assessed by event-free survival and stringent objective response measures. RESULTS: PI3Kδ and PI3Kγ mRNA expression was significantly higher in B-lineage than T-lineage ALL PDXs (p-values <.0001). Duvelisib was well-tolerated and reduced leukemia cells in the peripheral blood in four PDXs, but with only one objective response. There was no obvious relationship between duvelisib efficacy and PI3Kδ or PI3Kγ expression or mutation status, nor was the in vivo response to duvelisib subtype dependent. CONCLUSIONS: Duvelisib demonstrated limited in vivo activity against ALL PDXs.

A novel and efficient tandem CD19- and CD22-directed CAR for B cell ALL.

CD19-directed chimeric antigen receptor (CAR) T cells have yielded impressive response rates in refractory/relapse B cell acute lymphoblastic leukemia (B-ALL); however, most patients ultimately relapse due to poor CAR T cell persistence or resistance of either CD19+ or CD19- B-ALL clones. CD22 is a pan-B marker whose expression is maintained in both CD19+ and CD19- relapses. CD22-CAR T cells have been clinically used in B-ALL patients, although relapse also occurs. T cells engineered with a tandem CAR (Tan-CAR) containing in a single construct both CD19 and CD22 scFvs may be advantageous in achieving higher remission rates and/or preventing antigen loss. We have generated and functionally validated using cutting-edge assays a 4-1BB-based CD22/CD19 Tan-CAR using in-house-developed novel CD19 and CD22 scFvs. Tan-CAR-expressing T cells showed similar in vitro expansion to CD19-CAR T cells with no increase in tonic signaling. CRISPR-Cas9-edited B-ALL cells confirmed the bispecificity of the Tan-CAR. Tan-CAR was as efficient as CD19-CAR in vitro and in vivo using B-ALL cell lines, patient samples, and patient-derived xenografts (PDXs). Strikingly, the robust antileukemic activity of the Tan-CAR was slightly more effective in controlling the disease in long-term follow-up PDX models. This Tan-CAR construct warrants a clinical appraisal to test whether simultaneous targeting of CD19 and CD22 enhances leukemia eradication and reduces/delays relapse rates and antigen loss.

An oncogene addiction phosphorylation signature and its derived scores inform tumor responsiveness to targeted therapies.

PURPOSE: Oncogene addiction provides important therapeutic opportunities for precision oncology treatment strategies. To date the cellular circuitries associated with driving oncoproteins, which eventually establish the phenotypic manifestation of oncogene addiction, remain largely unexplored. Data suggest the DNA damage response (DDR) as a central signaling network that intersects with pathways associated with deregulated addicting oncoproteins with kinase activity in cancer cells. EXPERIMENTAL: DESIGN: We employed a targeted mass spectrometry approach to systematically explore alterations in 116 phosphosites related to oncogene signaling and its intersection with the DDR following inhibition of the addicting oncogene alone or in combination with irradiation in MET-, EGFR-, ALK- or BRAF (V600)-positive cancer models. An NSCLC tissue pipeline combining patient-derived xenografts (PDXs) and ex vivo patient organotypic cultures has been established for treatment responsiveness assessment. RESULTS: We identified an 'oncogene addiction phosphorylation signature' (OAPS) consisting of 8 protein phosphorylations (ACLY S455, IF4B S422, IF4G1 S1231, LIMA1 S490, MYCN S62, NCBP1 S22, P3C2A S259 and TERF2 S365) that are significantly suppressed upon targeted oncogene inhibition solely in addicted cell line models and patient tissues. We show that the OAPS is present in patient tissues and the OAPS-derived score strongly correlates with the ex vivo responses to targeted treatments. CONCLUSIONS: We propose a score derived from OAPS as a quantitative measure to evaluate oncogene addiction of cancer cell samples. This work underlines the importance of protein phosphorylation assessment for patient stratification in precision oncology and corresponding identification of tumor subtypes sensitive to inhibition of a particular oncogene.

Thirteenth Annual ENBDC Workshop: Methods in Mammary Gland Biology and Breast Cancer.

The thirteenth annual workshop of the European Network for Breast Development and Cancer (ENBDC) Laboratories Annual Workshop took place on the 28-30 April 2022 in Weggis, Switzerland and focused on methods in mammary gland biology and breast cancer. Sixty scientists participated in the ENBDC annual workshop which had not been held in person since 2019 due to the global COVID-19 pandemic. Topics spanned the mammary gland biology field, ranging from lactation biology and embryonic development, single cell sequencing of the human breast, and stunning cutting-edge imaging of the mouse mammary gland and human breast as well as breast cancer research topics including invasive progression of the pre-invasive DCIS stage, metabolic determinants of endocrine therapy resistance, models for lobular breast cancer, and how mutational landscapes of normal breast during age and pregnancy determine cancer risk. The latest findings from participating researchers were presented through oral presentations and poster sessions and included plenty of unpublished work.

Deep whole genome sequencing identifies recurrent genomic alterations in commonly used breast cancer cell lines and patient-derived xenograft models.

BACKGROUND: Breast cancer cell lines (BCCLs) and patient-derived xenografts (PDXs) are the most frequently used models in breast cancer research. Despite their widespread usage, genome sequencing of these models is incomplete, with previous studies only focusing on targeted gene panels, whole exome or shallow whole genome sequencing. Deep whole genome sequencing is the most sensitive and accurate method to detect single nucleotide variants and indels, gene copy number and structural events such as gene fusions. RESULTS: Here we describe deep whole genome sequencing (WGS) of commonly used BCCL and PDX models using the Illumina X10 platform with an average ~ 60 × coverage. We identify novel genomic alterations, including point mutations and genomic rearrangements at base-pair resolution, compared to previously available sequencing data. Through integrative analysis with publicly available functional screening data, we annotate new genomic features likely to be of biological significance. CSMD1, previously identified as a tumor suppressor gene in various cancer types, including head and neck, lung and breast cancers, has been identified with deletion in 50% of our PDX models, suggesting an important role in aggressive breast cancers. CONCLUSIONS: Our WGS data provides a comprehensive genome sequencing resource of these models.

Palbociclib-based high-throughput combination drug screening identifies synergistic therapeutic options in HPV-negative head and neck squamous cell carcinoma.

BACKGROUND: Deregulation of cell-cycle pathway is ubiquitously observed in human papillomavirus negative (HPVneg) head and neck squamous cell carcinoma (HNSCC). Despite being an attractive target, CDK4/6 inhibition using palbociclib showed modest or conflicting results as monotherapy or in combination with platinum-based chemotherapy or cetuximab in HPVneg HNSCC. Thus, innovative agents to augment the efficacy of palbociclib in HPVneg HNSCC would be welcomed. METHODS: A collection of 162 FDA-approved and investigational agents was screened in combinatorial matrix format, and top combinations were validated in a broader panel of HPVneg HNSCC cell lines. Transcriptional profiling was conducted to explore the molecular mechanisms of drug synergy. Finally, the most potent palbociclib-based drug combination was evaluated and compared with palbociclib plus cetuximab or cisplatin in a panel of genetically diverse HPVneg HNSCC cell lines and patient-derived xenograft models. RESULTS: Palbociclib displayed limited efficacy in HPVneg HNSCC as monotherapy. The high-throughput combination drug screening provided a comprehensive palbociclib-based drug-drug interaction dataset, whereas significant synergistic effects were observed when palbociclib was combined with multiple agents, including inhibitors of the PI3K, EGFR, and MEK pathways. PI3K pathway inhibitors significantly reduced cell proliferation and induced cell-cycle arrest in HPVneg HNSCC cell lines when combined with palbociclib, and alpelisib (a PI3Kα inhibitor) was demonstrated to show the most potent synergy with particularly higher efficacy in HNSCCs bearing PIK3CA alterations. Notably, when compared with cisplatin and cetuximab, alpelisib exerted stronger synergism in a broader panel of cell lines. Mechanistically, RRM2-dependent epithelial mesenchymal transition (EMT) induced by palbociclib, was attenuated by alpelisib and cetuximab rather than cisplatin. Subsequently, PDX models with distinct genetic background further validated that palbociclib plus alpelisib had significant synergistic effects in models harboring PIK3CA amplification. CONCLUSIONS: This study provides insights into the systematic combinatory effect associated with CDK4/6 inhibition and supports further initiation of clinical trials using the palbociclib plus alpelisib combination in HPVneg HNSCC with PIK3CA alterations.

Longitudinal evaluation of five nasopharyngeal carcinoma animal models on the microPET/MR platform.

PURPOSE: We longitudinally evaluated the tumour growth and metabolic activity of three nasopharyngeal carcinoma (NPC) cell line models (C666-1, C17 and NPC43) and two xenograft models (Xeno76 and Xeno23) using a micropositron emission tomography and magnetic resonance (microPET/MR). With a better understanding of the interplay between tumour growth and metabolic characteristics of these NPC models, we aim to provide insights for the selection of appropriate NPC cell line/xenograft models to assist novel drug discovery and evaluation. METHODS: Mice were imaged by 18F-deoxyglucose ([18F]FDG) microPET/MR twice a week for consecutive 3-7 weeks. [18F]FDG uptake was quantified by standardized uptake value (SUV) and presented as SUVmean tumour-to-liver ratio (SUVRmean). Longitudinal tumour growth patterns and metabolic patterns were recorded. SUVRmean and histological characteristics were compared across the five NPC models. Cisplatin was administrated to one selected optimal tumour model, C17, to evaluate our imaging platform. RESULTS: We found variable tumour growth and metabolic patterns across different NPC tumour types. C17 has an optimal growth rate and higher tumour metabolic activity compared with C666-1. C666-1 has a fast growth rate but is low in SUVRmean at endpoint due to necrosis as confirmed by H&E. NPC43 and Xeno76 have relatively slow growth rates and are low in SUVRmean, due to severe necrosis. Xeno23 has the slowest growth rate, and a relative high SUVRmean. Cisplatin showed the expected therapeutic effect in the C17 model in marked reduction of tumour size and metabolism. CONCLUSION: Our study establishes an imaging platform that characterizes the growth and metabolic patterns of different NPC models, and the platform is well able to demonstrate drug treatment outcome supporting its use in novel drug discovery and evaluation for NPC.

B7-H3 targeted CAR-T cells show highly efficient anti-tumor function against osteosarcoma both in vitro and in vivo.

BACKGROUND: Osteosarcoma (OS) mainly happens in children and youths. Surgery, radiotherapy and chemotherapy are the common therapies for osteosarcoma treatment but all their anti-tumor effects are limited. In recent years, a new cellular therapy, CAR-T, a cellular immunotherapy with genetically engineered T cells bearing chimeric antigen receptor targeting specific tumor-associated antigen, has been proved to be an effective therapy against acute lymphoblastic leukemia. Thus, CAR-T is a potentially effective therapy for osteosarcoma treatment. METHODS: A CAR gene targeting B7-H3 antigen was constructed into lentiviral vector through molecular biology techniques. Then, the CAR gene was transferred to T cells through lentiviral delivery system, and the CAR-T cells were largely expanded using in vitro culture technology. The in vitro anti-tumor effect of CAR-T cells was evaluated through Real Time Cell Analysis system (RTCA) and ELISA assay. The in vivo anti-tumor capabilities of CAR-T cells were evaluated using the patient-derived xenografts (PDX) model of osteosarcoma. RESULTS: The third-generation CAR-T cells we constructed could target the B7-H3 antigen, and the phenotype of CAR-T cells was consistent with normal T cells; The CAR-T cells showed superior antitumor effects both in vitro and in vivo. CONCLUSION: Our study showed that B7-H3 targeted CAR-T cells had high anti-tumor efficacy against osteosarcoma both in vitro and in vivo, which proved that B7-H3 targeted CAR-T therapy is potentially effective for osteosarcoma treatment.

Patient-Derived In Vitro and In Vivo Models of Cancer.

Over the last two decades, cancer researchers have taken the promise offered by the Human Genome Project and have expanded its capacity to use sequencing to identify the genomic alterations that give rise to and sustain individual tumors. This expansion has allowed researchers to identify and target highly recurrent alterations in specific cancer contexts, such as EGFR mutations in non-small cell lung cancer (Lynch et al, N Engl J Med 350:2129-2139, 2004; Sharifnia et al., Proc Natl Acad Sci U S A 111:18661-18666, 2014), BCR-ABL translocations in chronic myeloid leukemia (Deininger, Pharmacol Rev 55:401-423. https://doi.org/10.1124/pr.55.3.4 , 2003; Druker et al, N Engl J Med 344. 1038-1042, 2001; Druker et al, N Engl J Med 344:1031-1037. https://doi.org/10.1056/NEJM200104053441401 , 2001), or HER2 amplifications in breast cancer (Slamon et al, N Engl J Med 344:783-792. https://doi.org/10.1056/NEJM200103153441101 , 2001; Solca et al, Beyond trastuzumab: second-generation targeted therapies for HER-2-positive breast cancer. In: Sibilia M, Zielinski CC, Bartsch R, Grunt TW (eds) Drugs for HER-2-positive breast cancer. Springer, Basel, pp 91-107, 2011). Despite these advances in our capacity to identify the genetic alterations that drive tumor initiation, survival, and proliferation, our ability to target these alterations to provide effective treatment options for patients in need, particularly those with rare or advanced cancers, remains limited (Gould et al, Nat Med 21:431-439. https://doi.org/10.1038/nm.3853 , 2015). Patient-derived models of cancer offer one potential mechanism to overcome this barrier between the bench and bedside. Through the development and testing of patient-derived models of cancer, functional genomics efforts can identify tumor-specific drug sensitivities and thereby provide a connection between tumor genetics and effective therapeutics for patients in need of treatment options.Recognizing that cancer is a multifaceted set of disease states, the development of personalized models of cancer that can be used to compare treatment options, identify tumor-specific vulnerabilities, and guide clinical decision-making has tremendous potential for improving patient outcomes. This chapter will describe a representative set of patient-derived models of cancer, reviewing each of their strengths and weaknesses and highlighting how selecting a model to suit a specific question or context is critical. Each model comes with a unique set of pros and cons, making them more or less appropriate for each specific research or clinical question. As each model can be leveraged to gain new insights into cancer biology, the key to their deployment is to identify the most appropriate model for a specific context, while carefully considering the strengths and limitations of the selected model. When used appropriately, patient-derived models may prove to be the missing link needed to bring the promise of personalized oncology to fruition in the clinic.

Enapotamab Vedotin, an AXL-Specific Antibody-Drug Conjugate, Demonstrates Antitumor Efficacy in Patient-Derived Xenograft Models of Soft Tissue Sarcoma.

Doxorubicin (doxo) remains the standard of care for patients with advanced soft tissue sarcoma (STS), even though response rates to doxo are only around 14% to 18%. We evaluated enapotamab vedotin (EnaV), an AXL-specific antibody-drug conjugate (ADC), in a panel of STS patient-derived xenografts (PDX). Eight models representing multiple STS subtypes were selected from our STS PDX platform (n = 45) by AXL immunostaining on archived passages. Models were expanded by unilateral transplantation of tumor tissue into the left flank of 20 NMRI nu/nu mice. Once tumors were established, mice were randomized into an EnaV treatment group, or a group treated with isotype control ADC. Treatment efficacy was assessed by tumor volume evaluation, survival analysis, and histological evaluation of tumors, and associated with AXL expression. EnaV demonstrated significant tumor growth delay, regression, and/or prolonged survival compared to isotype control ADC in 5/8 STS PDX models investigated. Experimental passages of responding models were all found positive for AXL at varying levels, but no linear relationship could be identified between the level of expression and level of response to EnaV. One model was found negative for AXL on experimental passage and did not respond to EnaV. This study provides a preclinical rationale for the evaluation of AXL-targeting ADCs in the treatment of AXL-expressing sarcomas.

Developing 3D Organoid Raft Cultures from Patient-Derived Xenografts as Rapid Models to Screen Efficacy of Experimental Therapeutics.

Reliable preclinical models are needed for screening new cancer drugs. Thus, we developed an improved 3D tumor organoid model termed "organoid raft cultures" (ORCs). Development of ORCs involved culturing tumors ex vivo on collagen beds (boats) with grid supports to maintain their morphological structure. The ORCs were developed from patient-derived xenografts (PDXs) of colon cancers excised from immune-deficient mice (NOD/SCID/IL2Rgammanull). We utilized these new models to evaluate the efficacy of an investigational drug, Navitoclax (ABT-263). We tested the efficacy of ABT-263, an inhibitor of BCL-2 family proteins, in these ORCs derived from a PDX that showed high expression of antiapoptotic BCL2 family proteins (BCL-2, BCL-XL, and BCL-W). Hematoxylin and eosin staining evaluation of PDXs and corresponding ORCs indicated the retention of morphological and other histological integrity of ORCs. ORCs treated with ABT-263 showed decreased expression of antiapoptotic proteins (BCL2, BCL-XL and BCL-W) and increased proapoptotic proteins (BAX and PUMA), with concomitant activation of caspase 3. These studies support the usefulness of the ORCs, developed from PDXs, as an alternative to PDXs and as faster screening models.

Multicellular Modelling of Difficult-to-Treat Gastrointestinal Cancers: Current Possibilities and Challenges.

Cancers affecting the gastrointestinal system are highly prevalent and their incidence is still increasing. Among them, gastric and pancreatic cancers have a dismal prognosis (survival of 5-20%) and are defined as difficult-to-treat cancers. This reflects the urge for novel therapeutic targets and aims for personalised therapies. As a prerequisite for identifying targets and test therapeutic interventions, the development of well-established, translational and reliable preclinical research models is instrumental. This review discusses the development, advantages and limitations of both patient-derived organoids (PDO) and patient-derived xenografts (PDX) for gastric and pancreatic ductal adenocarcinoma (PDAC). First and next generation multicellular PDO/PDX models are believed to faithfully generate a patient-specific avatar in a preclinical setting, opening novel therapeutic directions for these difficult-to-treat cancers. Excitingly, future opportunities such as PDO co-cultures with immune or stromal cells, organoid-on-a-chip models and humanised PDXs are the basis of a completely new area, offering close-to-human models. These tools can be exploited to understand cancer heterogeneity, which is indispensable to pave the way towards more tumour-specific therapies and, with that, better survival for patients.