Patient-Derived Models of Cancer in the NCI PDMC Consortium: Selection, Pitfalls, and Practical Recommendations.

For over a century, early researchers sought to study biological organisms in a laboratory setting, leading to the generation of both in vitro and in vivo model systems. Patient-derived models of cancer (PDMCs) have more recently come to the forefront of preclinical cancer models and are even finding their way into clinical practice as part of functional precision medicine programs. The PDMC Consortium, supported by the Division of Cancer Biology in the National Cancer Institute of the National Institutes of Health, seeks to understand the biological principles that govern the various PDMC behaviors, particularly in response to perturbagens, such as cancer therapeutics. Based on collective experience from the consortium groups, we provide insight regarding PDMCs established both in vitro and in vivo, with a focus on practical matters related to developing and maintaining key cancer models through a series of vignettes. Although every model has the potential to offer valuable insights, the choice of the right model should be guided by the research question. However, recognizing the inherent constraints in each model is crucial. Our objective here is to delineate the strengths and limitations of each model as established by individual vignettes. Further advances in PDMCs and the development of novel model systems will enable us to better understand human biology and improve the study of human pathology in the lab.

Effects and Eradication of Mycoplasma Contamination on Patient-derived Colorectal Cancer Organoid Cultures.

Patient-derived organoids are a useful platform for identification and testing of novel precision oncology approaches. Patient-derived organoids are generated by direct culture of patient samples. However, prior to development into patient-derived organoids, these samples are often processed for clinical use, opening the potential for contamination by Mycoplasma and other microbes. While most microbes can be detected by visual inspection, Mycoplasma can go undetected and have substantial impacts on assay results. Given the increased use of patient-derived organoids, there is a growing need for a standardized protocol to detect and remove Mycoplasma from organoid models. In the current study, we report a procedure for Mycoplasma removal by passaging organoids through mice as patient-derived organoid xenografts. In vivo passage of patient-derived organoids followed by re-establishment was 100% effective at decontaminating colorectal patient-derived organoids (n = 9), based on testing with the Sigma LookOut Mycoplasma PCR Detection Kit. This process can serve as a method to re-establish contaminated patient-derived organoids, which represent precious models to study patient-specific genomic features and treatment responses. SIGNIFICANCE: Organoids are valuable models of cancer. Mycoplasma contamination can alter organoid drug sensitivity, so there is a need for a standardized protocol to detect and remove Mycoplasma from organoids. We report a simple procedure for removing Mycoplasma from organoids via in vivo passaging through mice followed by re-establishment of organoids.

Corrigendum: Psymberin, a marine-derived natural product, induces cancer cell growth arrest and protein translation inhibition.

[This corrects the article DOI: 10.3389/fmed.2022.999004.].

Protein Folding Stability Profiling of Colorectal Cancer Chemoresistance Identifies Functionally Relevant Biomarkers.

Reported here is the application of three protein folding stability profiling techniques (including the stability of proteins from rates of oxidation, thermal protein profiling, and limited proteolysis approaches) to identify differentially stabilized proteins in six patient-derived colorectal cancer (CRC) cell lines with different oxaliplatin sensitivities and eight CRC patient-derived xenografts (PDXs) derived from two of the patient derived cell lines with different oxaliplatin sensitivities. Compared to conventional protein expression level analyses, which were also performed here, the stability profiling techniques identified both unique and novel proteins and cellular components that differentiated the sensitive and resistant samples including 36 proteins that were differentially stabilized in at least two techniques in both the cell line and PDX studies of oxaliplatin resistance. These 36 differentially stabilized proteins included 10 proteins previously connected to cancer chemoresistance. Two differentially stabilized proteins, fatty acid synthase and elongation factor 2, were functionally validated in vitro and found to be druggable protein targets with biological functions that can be modulated to improve the efficacy of CRC chemotherapy. These results add to our understanding of CRC oxaliplatin resistance, suggest biomarker candidates for predicting oxaliplatin sensitivity in CRC, and inform new strategies for overcoming chemoresistance in CRC.

An autologous humanized patient-derived xenograft (PDX) model for evaluation of nivolumab immunotherapy in renal cell cancer: a case report.

Background: There is an unmet need for developing faithful animal models for preclinical evaluation of immunotherapy. The current approach to generate preclinical models for immunotherapy evaluation has been to transplant CD34+ cells from umbilical cord blood into immune-deficient mice followed by implantation of patient derived tumor cells. However, current models are associated with high tumor rejection rate secondary to the allograft vs. tumor response from human leukocyte antigen (HLA) mismatches. We herein report the first development of a novel, humanized patient-derived xenograft (PDX) model using autologous CD34+ cells from bone marrow aspirate obtained from a patient with metastatic clear cell renal cell carcinoma (mRCC) from whom a PDX had been developed. Case Description: This is a 68-year-old Caucasian man diagnosed with mRCC with metastasis to the liver in 2014. He was treated with sunitinib +/- AGS-003 and underwent a cytoreductive right nephrectomy, left adrenalectomy and partial liver resection. PDX was generated using resected nephrectomy specimen. After surgery, patient received multiple lines of standard of care therapy including sunitinib, axitinib, bevacizumab, everolimus and cabozantinib. While progressing on cabozantinib, he was treated with nivolumab. Seven years after initiation of nivolumab, and 4 years after stopping systemic therapy, he remains in complete remission. To generate autologous PDX model, bone marrow aspirate was performed and CD34+ hematopoietic stem/progenitor cells (HSPCs) were isolated and injected into 150 rad irradiated non-obese diabetic scid gamma null (NSG) mice. At 11 weeks post-transplant, the matched patient PDX was injected subcutaneously into the humanized mice and the mice were treated with nivolumab. Conclusions: Our case represents successful therapy of nivolumab in mRCC. Furthermore, HPSCs obtained from a single bone marrow aspirate were able to reconstitute an immune system in the mice that allowed nivolumab to inhibit the tumor growth of PDX and recapitulated the durable remission observed in the patient with nivolumab. We observed the reconstitution of human T cells, B cells and natural killer (NK) cells and unlike the humanized mouse model using cord blood, our model system eliminates the tumor rejection from mis-matched HLA. Our autologous humanized renal cell carcinoma (RCC) PDX model provides an effective tool to study immunotherapy in a preclinical setting.

Psymberin, a marine-derived natural product, induces cancer cell growth arrest and protein translation inhibition.

Colorectal cancer (CRC) is the third most prevalent form of cancer in the United States and results in over 50,000 deaths per year. Treatments for metastatic CRC are limited, and therefore there is an unmet clinical need for more effective therapies. In our prior work, we coupled high-throughput chemical screens with patient-derived models of cancer to identify new potential therapeutic targets for CRC. However, this pipeline is limited by (1) the use of cell lines that do not appropriately recapitulate the tumor microenvironment, and (2) the use of patient-derived xenografts (PDXs), which are time-consuming and costly for validation of drug efficacy. To overcome these limitations, we have turned to patient-derived organoids. Organoids are increasingly being accepted as a "standard" preclinical model that recapitulates tumor microenvironment cross-talk in a rapid, cost-effective platform. In the present work, we employed a library of natural products, intermediates, and drug-like compounds for which full synthesis has been demonstrated. Using this compound library, we performed a high-throughput screen on multiple low-passage cancer cell lines to identify potential treatments. The top candidate, psymberin, was further validated, with a focus on CRC cell lines and organoids. Mechanistic and genomics analyses pinpointed protein translation inhibition as a mechanism of action of psymberin. These findings suggest the potential of psymberin as a novel therapy for the treatment of CRC.

Characterization of a castrate-resistant prostate cancer xenograft derived from a patient of West African ancestry.

BACKGROUND: Prostate cancer is a clinically and molecularly heterogeneous disease, with highest incidence and mortality among men of African ancestry. To date, prostate cancer patient-derived xenograft (PCPDX) models to study this disease have been difficult to establish because of limited specimen availability and poor uptake rates in immunodeficient mice. Ancestrally diverse PCPDXs are even more rare, and only six PCPDXs from self-identified African American patients from one institution were recently made available. METHODS: In the present study, we established a PCPDX from prostate cancer tissue from a patient of estimated 90% West African ancestry with metastatic castration resistant disease, and characterized this model's pathology, karyotype, hotspot mutations, copy number, gene fusions, gene expression, growth rate in normal and castrated mice, therapeutic response, and experimental metastasis. RESULTS: This PCPDX has a mutation in TP53 and loss of PTEN and RB1. We have documented a 100% take rate in mice after thawing the PCPDX tumor from frozen stock. The PCPDX is castrate- and docetaxel-resistant and cisplatin-sensitive, and has gene expression patterns associated with such drug responses. After tail vein injection, the PCPDX tumor cells can colonize the lungs of mice. CONCLUSION: This PCPDX, along with others that are established and characterized, will be useful pre-clinically for studying the heterogeneity of prostate cancer biology and testing new therapeutics in models expected to be reflective of the clinical setting.

Circadian clock, carcinogenesis, chronochemotherapy connections.

The circadian clock controls the expression of nearly 50% of protein coding genes in mice and most likely in humans as well. Therefore, disruption of the circadian clock is presumed to have serious pathological effects including cancer. However, epidemiological studies on individuals with circadian disruption because of night shift or rotating shift work have produced contradictory data not conducive to scientific consensus as to whether circadian disruption increases the incidence of breast, ovarian, prostate, or colorectal cancers. Similarly, genetically engineered mice with clock disruption do not exhibit spontaneous or radiation-induced cancers at higher incidence than wild-type controls. Because many cellular functions including the cell cycle and cell division are, at least in part, controlled by the molecular clock components (CLOCK, BMAL1, CRYs, PERs), it has also been expected that appropriate timing of chemotherapy may increase the efficacy of chemotherapeutic drugs and ameliorate their side effect. However, empirical attempts at chronochemotherapy have not produced beneficial outcomes. Using mice without and with human tumor xenografts, sites of DNA damage and repair following treatment with the anticancer drug cisplatin have been mapped genome-wide at single nucleotide resolution and as a function of circadian time. The data indicate that mechanism-based studies such as these may provide information necessary for devising rational chronochemotherapy regimens.

Integrated chromatin and transcriptomic profiling of patient-derived colon cancer organoids identifies personalized drug targets to overcome oxaliplatin resistance.

Colorectal cancer is a leading cause of cancer deaths. Most colorectal cancer patients eventually develop chemoresistance to the current standard-of-care therapies. Here, we used patient-derived colorectal cancer organoids to demonstrate that resistant tumor cells undergo significant chromatin changes in response to oxaliplatin treatment. Integrated transcriptomic and chromatin accessibility analyses using ATAC-Seq and RNA-Seq identified a group of genes associated with significantly increased chromatin accessibility and upregulated gene expression. CRISPR/Cas9 silencing of fibroblast growth factor receptor 1 (FGFR1) and oxytocin receptor (OXTR) helped overcome oxaliplatin resistance. Similarly, treatment with oxaliplatin in combination with an FGFR1 inhibitor (PD166866) or an antagonist of OXTR (L-368,899) suppressed chemoresistant organoids. However, oxaliplatin treatment did not activate either FGFR1 or OXTR expression in another resistant organoid, suggesting that chromatin accessibility changes are patient-specific. The use of patient-derived cancer organoids in combination with transcriptomic and chromatin profiling may lead to precision treatments to overcome chemoresistance in colorectal cancer.

A Comparative Oncology Drug Discovery Pipeline to Identify and Validate New Treatments for Osteosarcoma.

BACKGROUND: Osteosarcoma is a rare but aggressive bone cancer that occurs primarily in children. Like other rare cancers, treatment advances for osteosarcoma have stagnated, with little improvement in survival for the past several decades. Developing new treatments has been hampered by extensive genomic heterogeneity and limited access to patient samples to study the biology of this complex disease. METHODS: To overcome these barriers, we combined the power of comparative oncology with patient-derived models of cancer and high-throughput chemical screens in a cross-species drug discovery pipeline. RESULTS: Coupling in vitro high-throughput drug screens on low-passage and established cell lines with in vivo validation in patient-derived xenografts we identify the proteasome and CRM1 nuclear export pathways as therapeutic sensitivities in osteosarcoma, with dual inhibition of these pathways inducing synergistic cytotoxicity. CONCLUSIONS: These collective efforts provide an experimental framework and set of new tools for osteosarcoma and other rare cancers to identify and study new therapeutic vulnerabilities.

A Precision Medicine Drug Discovery Pipeline Identifies Combined CDK2 and 9 Inhibition as a Novel Therapeutic Strategy in Colorectal Cancer.

Colorectal cancer is the third most common cancer in the United States and responsible for over 50,000 deaths each year. Therapeutic options for advanced colorectal cancer are limited, and there remains an unmet clinical need to identify new treatments for this deadly disease. To address this need, we developed a precision medicine pipeline that integrates high-throughput chemical screens with matched patient-derived cell lines and patient-derived xenografts (PDX) to identify new treatments for colorectal cancer. High-throughput screens of 2,100 compounds were performed across six low-passage, patient-derived colorectal cancer cell lines. These screens identified the CDK inhibitor drug class among the most effective cytotoxic compounds across six colorectal cancer lines. Among this class, combined targeting of CDK1, 2, and 9 was the most effective, with IC50s ranging from 110 nmol/L to 1.2 µmol/L. Knockdown of CDK9 in the presence of a CDK2 inhibitor (CVT-313) showed that CDK9 knockdown acted synergistically with CDK2 inhibition. Mechanistically, dual CDK2/9 inhibition induced significant G2-M arrest and anaphase catastrophe. Combined CDK2/9 inhibition in vivo synergistically reduced PDX tumor growth. Our precision medicine pipeline provides a robust screening and validation platform to identify promising new cancer therapies. Application of this platform to colorectal cancer pinpointed CDK2/9 dual inhibition as a novel combinatorial therapy to treat colorectal cancer.

An Aptamer for Broad Cancer Targeting and Therapy.

Recent advances in chemotherapy treatments are increasingly targeted therapies, with the drug conjugated to an antibody able to deliver it directly to the tumor. As high-affinity chemical ligands that are much smaller in size, aptamers are ideal for this type of drug targeting. Aptamer-highly toxic drug conjugates (ApTDCs) based on the E3 aptamer, selected on prostate cancer cells, target and inhibit prostate tumor growth in vivo. Here, we observe that E3 also broadly targets numerous other cancer types, apparently representing a universal aptamer for cancer targeting. Accordingly, ApTDCs formed by conjugation of E3 to the drugs monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF) efficiently target and kill a range of different cancer cells. Notably, this targeting extends to both patient-derived explant (PDX) cancer cell lines and tumors, with the E3 MMAE and MMAF conjugates inhibiting PDX cell growth in vitro and with the E3 aptamer targeting PDX colorectal tumors in vivo.

Development of a precision medicine pipeline to identify personalized treatments for colorectal cancer.

BACKGROUND: Metastatic colorectal cancer (CRC) continues to be a major health problem, and current treatments are primarily for disease control and palliation of symptoms. In this study, we developed a precision medicine strategy to discover novel therapeutics for patients with CRC. METHODS: Six matched low-passage cell lines and patient-derived xenografts (PDX) were established from CRC patients undergoing resection of their cancer. High-throughput drug screens using a 119 FDA-approved oncology drug library were performed on these cell lines, which were then validated in vivo in matched PDXs. RNA-Seq analysis was then performed to identify predictors of response. RESULTS: Our study revealed marked differences in response to standard-of-care agents across patients and pinpointed druggable pathways to treat CRC. Among these pathways co-targeting of fibroblast growth factor receptor (FGFR), SRC, platelet derived growth factor receptor (PDGFR), or vascular endothelial growth factor receptor (VEGFR) signaling was found to be an effective strategy. Molecular analyses revealed potential predictors of response to these druggable pathways. CONCLUSIONS: Our data suggests that the use of matched low-passage cell lines and PDXs is a promising strategy to identify new therapies and pathways to treat metastatic CRC.

Preclinical Testing of a Novel Niclosamide Stearate Prodrug Therapeutic (NSPT) Shows Efficacy Against Osteosarcoma.

Therapeutic advances for osteosarcoma have stagnated over the past several decades, leading to an unmet clinical need for patients. The purpose of this study was to develop a novel therapy for osteosarcoma by reformulating and validating niclosamide, an established anthelminthic agent, as a niclosamide stearate prodrug therapeutic (NSPT). We sought to improve the low and inefficient clinical bioavailability of oral dosing, especially for the relatively hydrophobic classes of anticancer drugs. Nanoparticles were fabricated by rapid solvent shifting and verified using dynamic light scattering and UV-vis spectrophotometry. NSPT efficacy was then studied in vitro for cell viability, cell proliferation, and intracellular signaling by Western blot analysis; ex vivo pulmonary metastatic assay model; and in vivo pharmacokinetic and lung mouse metastatic model of osteosarcoma. NSPT formulation stabilizes niclosamide stearate against hydrolysis and delays enzymolysis; increases circulation in vivo with t 1/2 approximately 5 hours; reduces cell viability and cell proliferation in human and canine osteosarcoma cells in vitro at 0.2-2 µmol/L IC50; inhibits recognized growth pathways and induces apoptosis at 20 µmol/L; eliminates metastatic lesions in the ex vivo lung metastatic model; and when injected intravenously at 50 mg/kg weekly, it prevents metastatic spread in the lungs in a mouse model of osteosarcoma over 30 days. In conclusion, niclosamide was optimized for preclinical drug delivery as a unique prodrug nanoparticle injected intravenously at 50 mg/kg (1.9 mmol/L). This increased bioavailability of niclosamide in the blood stream prevented metastatic disease in the mouse. This chemotherapeutic strategy is now ready for canine trials, and if successful, will be targeted for human trials in patients with osteosarcoma.

Genome-wide single-nucleotide resolution of oxaliplatin-DNA adduct repair in drug-sensitive and -resistant colorectal cancer cell lines.

Platinum-based chemotherapies, including oxaliplatin, are a mainstay in the management of solid tumors and induce cell death by forming intrastrand dinucleotide DNA adducts. Despite their common use, they are highly toxic, and approximately half of cancer patients have tumors that are either intrinsically resistant or develop resistance. Previous studies suggest that this resistance is mediated by variations in DNA repair levels or net drug influx. Here, we aimed to better define the roles of nucleotide excision repair and DNA damage in platinum chemotherapy resistance by profiling DNA damage and repair efficiency in seven oxaliplatin-sensitive and three oxaliplatin-resistant colorectal cancer cell lines. We assayed DNA repair indirectly as toxicity and directly measured bulky adduct formation and removal from the genome by slot blot and repair capacity in an excision assay, and used excision repair sequencing (XR-seq) to map repair events genome-wide at single-nucleotide resolution. Using this combinatorial approach and proxies for oxaliplatin-DNA damage, we observed no significant differences in repair efficiency that could explain the relative sensitivities and chemotherapy resistances of these cell lines. In contrast, the levels of oxaliplatin-induced DNA damage were significantly lower in the resistant cells, indicating that decreased damage formation, rather than increased damage repair, is a major determinant of oxaliplatin resistance in these cell lines. XR-seq-based analysis of gene expression revealed up-regulation of membrane transport pathways in the resistant cells, and these pathways may contribute to resistance. In conclusion, additional research is needed to characterize the factors mitigating cellular DNA damage formation by platinum compounds.

Single-Cell RNA Sequencing Identifies Yes-Associated Protein 1-Dependent Hepatic Mesothelial Progenitors in Fibrolamellar Carcinoma.

Fibrolamellar carcinoma (FLC) is characterized by in-frame fusion of DnaJ heat shock protein family (Hsp40) member B1 (DNAJB1) with protein kinase cAMP-activated catalytic subunit α (PRKACA) and by dense desmoplasia. Surgery is the only effective treatment because mechanisms supporting tumor survival are unknown. We used single-cell RNA sequencing to characterize a patient-derived FLC xenograft model and identify therapeutic targets. Human FLC cells segregated into four discrete clusters that all expressed the oncogene Yes-associated protein 1 (YAP1). The two communities most enriched with cells coexpressing FLC markers [CD68, A-kinase anchoring protein 12 (AKAP12), cytokeratin 7, epithelial cell adhesion molecule (EPCAM), and carbamoyl palmitate synthase-1] also had the most cells expressing YAP1 and its proproliferative target genes (AREG and CCND1), suggesting these were proliferative FLC cell clusters. The other two clusters were enriched with cells expressing profibrotic YAP1 target genes, ACTA2, ELN, and COL1A1, indicating these were fibrogenic FLC cells. All clusters expressed the YAP1 target gene and mesothelial progenitor marker mesothelin, and many mesothelin-positive cells coexpressed albumin. Trajectory analysis predicted that the four FLC communities were derived from a single cell type transitioning among phenotypic states. After establishing a novel FLC cell line that harbored the DNAJB1-PRKACA fusion, YAP1 was inhibited, which significantly reduced expression of known YAP1 target genes as well as cell growth and migration. Thus, both FLC epithelial and stromal cells appear to arise from DNAJB1-PRKACA fusion in a YAP1-dependent liver mesothelial progenitor, identifying YAP1 as a target for FLC therapy.

Dietary methionine influences therapy in mouse cancer models and alters human metabolism.

Nutrition exerts considerable effects on health, and dietary interventions are commonly used to treat diseases of metabolic aetiology. Although cancer has a substantial metabolic component1, the principles that define whether nutrition may be used to influence outcomes of cancer are unclear2. Nevertheless, it is established that targeting metabolic pathways with pharmacological agents or radiation can sometimes lead to controlled therapeutic outcomes. By contrast, whether specific dietary interventions can influence the metabolic pathways that are targeted in standard cancer therapies is not known. Here we show that dietary restriction of the essential amino acid methionine-the reduction of which has anti-ageing and anti-obesogenic properties-influences cancer outcome, through controlled and reproducible changes to one-carbon metabolism. This pathway metabolizes methionine and is the target of a variety of cancer interventions that involve chemotherapy and radiation. Methionine restriction produced therapeutic responses in two patient-derived xenograft models of chemotherapy-resistant RAS-driven colorectal cancer, and in a mouse model of autochthonous soft-tissue sarcoma driven by a G12D mutation in KRAS and knockout of p53 (KrasG12D/+;Trp53-/-) that is resistant to radiation. Metabolomics revealed that the therapeutic mechanisms operate via tumour-cell-autonomous effects on flux through one-carbon metabolism that affects redox and nucleotide metabolism-and thus interact with the antimetabolite or radiation intervention. In a controlled and tolerated feeding study in humans, methionine restriction resulted in effects on systemic metabolism that were similar to those obtained in mice. These findings provide evidence that a targeted dietary manipulation can specifically affect tumour-cell metabolism to mediate broad aspects of cancer outcome.

Exercise inhibits tumor growth and central carbon metabolism in patient-derived xenograft models of colorectal cancer.

BACKGROUND: While self-reported exercise is associated with a reduction in the risk of recurrence in colorectal cancer, the molecular mechanisms underpinning this relationship are unknown. Furthermore, the effect of exercise on intratumoral metabolic processes has not been investigated in detail in human cancers. In our current study, we generated six colorectal patient patient-derived xenografts (CRC PDXs) models and treated each PDX to voluntary wheel running (exercise) for 6-8 weeks or no exposure to the wheel (control). A comprehensive metabolomics analysis was then performed on the PDXs to identify exercise induced changes in the tumor that were associated with slower growth. RESULTS: Tumor growth inhibition was observed in the voluntary wheel running group compared to the control group in three of the six models. A metabolomics analysis first revealed that central carbon metabolism was affected in each model irrespective of treatment. Interestingly, comparison of responsive and resistant models showed that levels of metabolites in nucleotide metabolism, known to be coupled to mitochondrial metabolism, were predictive of response. Furthermore, phosphocreatine levels which are linked to mitochondrial energy demands were associated with inhibition of tumor growth. CONCLUSION: Altogether, this study provides evidence that changes to tumor cell mitochondrial metabolism may underlie in part the benefits of exercise.

Single-Cell Transcriptomics Reveals Heterogeneity and Drug Response of Human Colorectal Cancer Organoids.

Organoids are three-dimensional cell cultures that mimic organ functions and structures. The organoid model has been developed as a versatile in vitro platform for stem cell biology and diseases modeling. Tumor organoids are shown to share ~ 90% of genetic mutations with biopsies from same patients. However, it's not clear whether tumor organoids recapitulate the cellular heterogeneity observed in patient tumors. Here, we used single-cell RNA-Seq to investigate the transcriptomics of tumor organoids derived from human colorectal tumors, and applied machine learning methods to unbiasedly cluster subtypes in tumor organoids. Computational analysis reveals cancer heterogeneity sustained in tumor organoids, and the subtypes in organoids displayed high diversity. Furthermore, we treated the tumor organoids with a first-line cancer drug, Oxaliplatin, and investigated drug response in single-cell scale. Diversity of tumor cell populations in organoids were significantly perturbed by drug treatment. Single-cell analysis detected the depletion of chemosensitive subgroups and emergence of new drug tolerant subgroups after drug treatment. Our study suggests that the organoid model is capable of recapitulating clinical heterogeneity and its evolution in response to chemotherapy.

Targeting HPV16 DNA using CRISPR/Cas inhibits anal cancer growth in vivo.

AIM: The goal of this study was to determine if a single AAV vector, encoding Cas9 and guide RNAs specific for the HPV16 E6 and E7 genes, could inhibit the growth of an HPV16-induced tumor in vivo. MATERIALS & METHODS: We grew HPV16+, patient-derived anal cancer explants in immunodeficient mice and then challenged these by injection of AAV-based vectors encoding Cas9 and control or HPV16-specific guide RNAs. RESULTS & CONCLUSION: We observed a significant and selective reduction in tumor growth when the HPV16 E6 and E7 genes were targeted using Cas9. These studies provide proof of principle for the hypothesis that CRISPR/Cas has the potential to be used to selectively treat HPV-induced tumors in humans.