Host Mice Research Articles

Human cerebral organoids establish subcortical projections in the mouse brain after transplantation

Numerous studies have used human pluripotent stem cell-derived cerebral organoids to elucidate the mystery of human brain development and model neurological diseases in vitro, but the potential for grafted organoid-based therapy in vivo remains unknown. Here, we optimized a culturing protocol capable of efficiently generating small human cerebral organoids. After transplantation into the mouse medial prefrontal cortex, the grafted human cerebral organoids survived and extended projections over 4.5 mm in length to basal brain regions within 1 month. The transplanted cerebral organoids generated human glutamatergic neurons that acquired electrophysiological maturity in the mouse brain. Importantly, the grafted human cerebral organoids functionally integrated into pre-existing neural circuits by forming bidirectional synaptic connections with the mouse host neurons. Furthermore, compared to control mice, the mice transplanted with cerebral organoids showed an increase in freezing time in response to auditory conditioned stimuli, suggesting the potentiation of the startle fear response. Our study showed that subcortical projections can be established by microtransplantation and may provide crucial insights into the therapeutic potential of human cerebral organoids for neurological diseases.

IRF4 Expression Is Required for the Immunoregulatory Activity of Conventional Type 2 Dendritic Cells in Settings of Chronic Bacterial Infection and Cancer.

The lamina propria of the gastrointestinal tract and other mucosal surfaces of humans and mice host a network of mononuclear phagocytes that differ in their ontogeny, surface marker and transcription factor expression, and functional specialization. Conventional dendritic cells (DCs) in particular exist as two major subpopulations in both lymphoid and nonlymphoid organs that can be distinguished based on their surface marker and transcription factor expression. In this study, we show in various Th1- and/or Th17-polarized settings of acute and chronic bacterial infection and of tumor growth that the conditional ablation of Irf4 in CD11c+ DCs results in more efficient immune control of Helicobacter pylori, Mycobacterium bovis bacillus Calmette-Guérin, and Citrobacter rodentium and of tumor growth in a syngeneic tumor model. We attribute the phenotype of IRF4ΔDC mice to unrestricted Th1 responses and in particular to IFN-γ- and TNF-α-expressing CD4+ T cells. This activity of IRF4-expressing DCs is linked to a DC-specific immunoregulatory transcriptional program. In contrast, in Th2-polarized settings such as house dust mite-induced allergic airway inflammation, the lack of IRF4 expression in the DC compartment alleviates inflammation and goblet cell metaplasia. The combined data provide evidence for immunoregulatory properties of this versatile DC population in Th1-polarized infection settings.

Fertility Preservation in Childhood Cancer: Endocrine Activity in Prepubertal Human Testis Xenografts Exposed to a Pubertal Hormone Environment.

Simple SummarySubstantial strides have been made in treating childhood cancers; however, as a result of chemotherapy and radiotherapy, young males experience long-term side effects, including impaired fertility. Whilst prepubertal testicular tissue can be cryopreserved prior to gonadotoxic treatments, it remains to be determined how to generate mature gametes from the immature human testis tissue. Development of immature germ cells into sperm is a complex process, which is supported by mature Sertoli cells and testosterone produced from Leydig cells. We used an established testicular xenotransplantation model to investigate the effect of puberty hormones, known as gonadotrophins, on functional maturation of the spermatogonial stem cell (SSC) niche. Limited testosterone production and partial maturation of Sertoli cells occurred in prepubertal testis grafts, suggesting that longer periods of grafting and/or identification of additional factors are required to develop testicular transplantation as a model for fertility preservation in male survivors of childhood cancer.Survivors of childhood cancer are at risk for long-term treatment-induced health sequelae, including gonadotoxicity and iatrogenic infertility. At present, for prepubertal boys there are no viable clinical options to preserve future reproductive potential. We investigated the effect of a pubertal induction regimen with gonadotrophins on prepubertal human testis xenograft development. Human testis tissue was obtained from patients with cancer and non-malignant haematological disorders (n = 6; aged 1–14 years) who underwent testis tissue cryopreservation for fertility preservation. Fresh and frozen-thawed testis fragments were transplanted subcutaneously or intratesticularly into immunocompromised mice. Graft-bearing mice received injections of vehicle or exogenous gonadotrophins, human chorionic gonadotrophin (hCG, 20 IU), and follicle-stimulating hormone (FSH, 12.5 IU) three times a week for 12 weeks. The gross morphology of vehicle and gonadotrophin-exposed grafts was similar for both transplantation sites. Exposure of prepubertal human testis tissue xenografts to exogenous gonadotrophins resulted in limited endocrine function of grafts, as demonstrated by the occasional expression of the steroidogenic cholesterol side-chain cleavage enzyme (CYP11A1). Plasma testosterone concentrations (0.13 vs. 0.25 ng/mL; p = 0.594) and seminal vesicle weights (10.02 vs. 13.93 mg; p = 0.431) in gonadotrophin-exposed recipient mice were comparable to vehicle-exposed controls. Regardless of the transplantation site and treatment, initiation and maintenance of androgen receptor (AR) expression were observed in Sertoli cells, indicating commitment towards a more differentiated status. However, neither exogenous gonadotrophins (in castrated host mice) nor endogenous testosterone (in intact host mice) were sufficient to repress the expression of markers associated with immature Sertoli cells, such as anti-Müllerian hormone (AMH) and Ki67, or to induce the redistribution of junctional proteins (connexin 43, CX43; claudin 11, CLDN11) to areas adjacent to the basement membrane. Spermatogonia did not progress developmentally but remained the most advanced germ cell type in testis xenografts. Overall, these findings demonstrate that exogenous gonadotrophins promote partial activation and maturation of the somatic environment in prepubertal testis xenografts. However, alternative hormone regimens or additional factors for pubertal induction are required to complete the functional maturation of the spermatogonial stem cell (SSC) niche.

Modulation of the Gut Microbiota Alters the Tumour-Suppressive Efficacy of Tim-3 Pathway Blockade in a Bacterial Species- and Host Factor-Dependent Manner.

T cell immunoglobulin and mucin domain-containing protein-3 (Tim-3) is an immune checkpoint molecule and a target for anti-cancer therapy. In this study, we examined whether gut microbiota manipulation altered the anti-tumour efficacy of Tim-3 blockade. The gut microbiota of mice was manipulated through the administration of antibiotics and oral gavage of bacteria. Alterations in the gut microbiome were analysed by 16S rRNA gene sequencing. Gut dysbiosis triggered by antibiotics attenuated the anti-tumour efficacy of Tim-3 blockade in both C57BL/6 and BALB/c mice. Anti-tumour efficacy was restored following oral gavage of faecal bacteria even as antibiotic administration continued. In the case of oral gavage of Enterococcus hirae or Lactobacillus johnsonii, transferred bacterial species and host mouse strain were critical determinants of the anti-tumour efficacy of Tim-3 blockade. Bacterial gavage did not increase the alpha diversity of gut microbiota in antibiotic-treated mice but did alter the microbiome composition, which was associated with the restoration of the anti-tumour efficacy of Tim-3 blockade. Conclusively, our results indicate that gut microbiota modulation may improve the therapeutic efficacy of Tim-3 blockade during concomitant antibiotic treatment. The administered bacterial species and host factors should be considered in order to achieve therapeutically beneficial modulation of the microbiota.

Generation of Human Renal Vesicles in Mouse Organ Niche Using Nephron Progenitor Cell Replacement System

Animal fetuses may be used for the regeneration of human organs. We have previously generated a transgenic mouse model that allows diphtheria toxin (DT)-induced ablation of Six2-positive nephron progenitor cells (NPCs). Elimination of existing native host NPCs enables their replacement with donor NPCs, which can generate neo-nephrons. However, this system cannot be applied to human NPCs, because DT induces apoptosis in human cells. Therefore, the present study presents a transgenic mouse model for the ablation of NPCs using tamoxifen, which does not affect human cells. Using this system, we successfully regenerate interspecies neo-nephrons, which exhibit urine-producing abilities, from transplanted rat NPCs in a mouse host. Transplantation of human induced pluripotent stem cell (iPSC)-derived NPCs results in differentiation into renal vesicles, which connect to the ureteric bud of the host. Thus, we demonstrate thepossibility of the regeneration of human kidneys derived from human iPSC-derived NPCs via NPC replacement.

P2X7 Receptor Promotes Mouse Mammary Cancer Cell Invasiveness and Tumour Progression, and Is a Target for Anticancer Treatment

The P2X7 receptor is an ATP-gated cation channel with a still ambiguous role in cancer progression, proposed to be either pro- or anti-cancerous, depending on the cancer or cell type in the tumour. Its role in mammary cancer progression is not yet defined. Here, we show that P2X7 receptor is functional in highly aggressive mammary cancer cells, and induces a change in cell morphology with fast F-actin reorganization and formation of filopodia, and promotes cancer cell invasiveness through both 2- and 3-dimensional extracellular matrices in vitro. Furthermore, P2X7 receptor sustains Cdc42 activity and the acquisition of a mesenchymal phenotype. In an immunocompetent mouse mammary cancer model, we reveal that the expression of P2X7 receptor in cancer cells, but not in the host mice, promotes tumour growth and metastasis development, which were reduced by treatment with specific P2X7 antagonists. Our results demonstrate that P2X7 receptor drives mammary tumour progression and represents a pertinent target for mammary cancer treatment.

Abstract 4029: BET inhibitor suppresses neutrophil-dependent lung metastasis of inflammatory renal cancer cells

Abstract Recent studies have shown that cancer cells alter the immune landscape by secreting inflammation-related cytokines or chemokines through a process known as cancer cell-intrinsic inflammation. Cancer cell-intrinsic inflammation facilitates both the initial progression of cancers and cancer metastasis via tumor microenvironment reconstitution, immune surveillance, and premetastatic niche formation. However, in the case of clear cell renal carcinoma (ccRCC), the role of cancer cell-intrinsic inflammation is not fully uncovered. In the present study, we explored the function and regulatory mechanisms of cancer cell-intrinsic inflammation in ccRCC. First, highly malignant derivatives were obtained through the repetitive exposure of human ccRCC cells into mouse renal microenvironment. Of note, these derivatives elicited cancer cell-intrinsic inflammation in the host mice, which facilitated lung metastasis in neutrophil-dependent manner. Thus, we defined these derivatives as inflammatory ccRCC cells and characterized in the following study. Next, the regulatory mechanisms for the transcription of genes related to neutrophil dynamics were explored. RNA-sequencing analysis revealed that mRNA-expression levels of four CXC chemokines were up-regulated in all inflammatory ccRCC derivatives. ChIP-sequencing analysis of H3K27ac or bromodomain-containing 4 (BRD4) indicated that super enhancers were recruited at each gene in inflammatory ccRCC derivatives. Finally, we evaluated the effect of the bromodomain and extra-terminal motif (BET) inhibitor on neutrophil-dependent lung metastasis of inflammatory ccRCC cells. Although the sizes of primary tumors in inflammatory ccRCC derivative-bearing mice were not altered by the treatment of BET inhibitor, those of metastatic lung tumors were significantly decreased. Overall, our findings revealed a functional role of cancer cell-intrinsic inflammation, which primes neutrophils and drives metastasis of ccRCC cells. The primary tumor microenvironment enriched inflammatory ccRCC cells via epigenetic remodeling. We also showed the utility of inflammation management in suppressing metastasis by utilizing a BET inhibitor. Citation Format: Shogo Ehata, Jun Nishida, Kosuke Miyakuni, Daizo Koinuma, Kohei Miyazono. BET inhibitor suppresses neutrophil-dependent lung metastasis of inflammatory renal cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4029.

Abstract 1673: Conservation of copy number profiles during engraftment and passaging of patient-derived cancer xenografts

Abstract Patient-derived xenografts (PDXs) are resected human tumors engrafted into mice for preclinical studies and therapeutic testing. It has been proposed that the mouse host affects tumor evolution during PDX engraftment and propagation, which could impact the capacity of PDXs for faithful modeling of patient treatment response. Such results contrast with reports that have observed genomic fidelity of PDX models with respect to the originating patient tumors and from early to late passages by direct DNA measurements (DNA sequencing or SNP arrays). Here we resolve these contradicting observations by systematically evaluating CNA changes and the genes they affect during engraftment and passaging in a large, internationally collected set of PDX models, comparing both RNA and DNA-based approaches. The data collected, as part of the U.S. National Cancer Institute (NCI) PDXNet (PDX Development and Trial Centers Research Network) Consortium and EurOPDX consortium, comprises 1548 patient (PT) and PDX datasets (1451 unique samples) from 509 models derived from American, European and Asian cancer patients, spanning across 16 tumor types. By assessing copy number changes by pairwise (PT-PDX or PDX-PDX) correlation and residual analysis to control for systematic biases, our study demonstrates that prior reports of systematic copy number divergence between PTs and PDXs are incorrect, and confirms the high retention of copy number during PDX engraftment and passaging. Moreover, only a small proportion of models show large CNA discordance between the samples, suggesting that the variations observed in PDX are mainly due to rare clonal selection of individual tumors rather than murine pressures. This large scale data analysis also reveals several other findings that clarify the evolutionary process in PDXs. We do observe larger deviations between PT-PDX than in PDX-PDX comparisons, likely due to dilution of PT signal by human stromal cells. Interestingly, we found that a major contributor to the differences between PDX samples is lineage-specific drift associated with splitting of tumors into fragments during PDX propagation. We observed no significant enrichment of cancer-related genes in PDX-specific CNAs across models, suggesting the lack of systematic copy number evolution driven by the PDX mouse host. Moreover, CNA differences between patient and PDX tumors were comparable to variations in multi-region tumor samples or intra-patient samples. Thus concerns about the genetic stability of the PDX system are likely to be less important than the spatial heterogeneity of solid tumors themselves. This result is consistent with our results on lineage effects during passaging, which indicate that intratumoral spatial evolution is the major reason for genetic drift. Our in-depth tracking of CNAs throughout PDX engraftment and passaging confirms that tumors engrafted and passaged in PDX models maintain a high degree of molecular fidelity to the original patient tumors and their suitability for pre-clinical drug testing. This work also finely enumerates the copy number profiles in hundreds of publicly available models, which will enable researchers to assess the suitability of each for individualized treatment studies. Citation Format: Xing Yi Woo, Jessica Giordano, Anuj Srivastava, Zi-Ming Zhao, Michael W. Lloyd, Roebi de Bruijn, Yun-Suhk Suh, Jong-Il Kim, Han-Kwang Yang, Charles Lee, Dennis A. Dean, Brandi Davis-Dusenbery, Yvonne A. Evrard, James H. Doroshow, Alana L. Welm, Bryan Welm, Michael T. Lewis, Bingliang Fang, Jack A. Roth, Funda Meric-Bernstam, Meenhard Herlyn, Michael Davies, Li Ding, Shunqiang Li, Ramaswamy Govindan, Claudio Isella, Jeffrey A. Moscow, Livio Trusolino, Annette Byrne, Jos Jonkers, Carol J. Bult, Enzo Medico, Jeffrey H. Chuang, PDXNET consortium & EurOPDX consortium. Conservation of copy number profiles during engraftment and passaging of patient-derived cancer xenografts [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1673.

Abstract 3130: Evaluation of TK1 protein changes using the AroCell TK 210 ELISA in an acute myeloid leukemia (AML) xenograft model during 5-fluoruracil treatment

Abstract Background Thymidine kinase 1 (TK1) is a cytosolic enzyme involved in DNA precursor synthesis and it is up-regulated in proliferating cells. Serum TK1 is an established biomarker for prognosis and therapy monitoring of malignancies. However, commercial TK1 enzyme activity assays have limitations for their use in xenograft models as they cross-react with TK1 activity from the host. The AroCell TK 210 ELISA is specific for human TK1 and offers the opportunity to measure the release of TK1 from human xenografts free from the background of TK1 release from the host (mouse). Here we studied the changes in serum TK1 protein levels using the AroCell TK 210 ELISA in a xenograft model during 5-fluorouracil (5FU) treatment of Acute Myeloid Leukemia (AML). Experimental Procedures Human MV-4-11 leukemic cells from ATCC were cultured with 95% O2 and 5% CO2 at 37°C and in IMD medium (Sigma) supplemented with 10% FBS (HI media) containing 4mmol/L L-glutamine, 5 ng/mL GM-CSF and 1% penicillin and streptomycin. The cells were harvested during exponential growth and injected subcutaneously into the left flank of athymic BALB/c nude mice 8-10 weeks old. The mice were divided into controls that received no therapy (n=8) and an active group receiving 5FU (n=8). The treatment was initiated when the average tumor volume reached 50-100 mm3. Tumor bearing mice were treated with PBS (control) or 5-FU (active group 10 mg/kg, once daily) for three weeks. Serum samples (70-80 µL) were collected before treatment initiation, during treatment (once weekly for 3 weeks) and a final termination. Serum samples were stored at -80°C and TK1 protein levels were determined with the AroCell TK 210 ELISA. All the animal experiments were performed at TheraIndx life sciences Pvt Ltd, India. Results The tumor volumes were measured post-implantation of cells every 3 days throughout the study. There was a significant difference (p<0.05) in the mean tumor volume between 5-FU treated (5-FU) (108.75± 36.3 mm3) and untreated controls (294.63± 50.17 mm3) at the end of treatment. There was a significant reduction in the tumor volume from 2nd week onwards in 5-FU treated mice up to termination (4th week) compared to control mice. TK1 protein concentrations increased significantly from the 2nd week onwards in 5-FU treated mice (0.42± 0.19 µg/L) in comparison to controls (0.1± 0.02 µg/L) in spite of the decrease in tumor volume in response to treatment. No cross-reactivity of TK 210 ELISA with mouse TK1 was observed. Conclusions This study is the first evaluation of TK 210 ELISA as a biomarker for monitoring therapy with anticancer drugs in an AML xenograft model. The changes in TK1 protein levels were significant enough to monitor the response of the xenograft to 5FU therapy in this animal model. Furthermore, the use of the AroCell TK 210 ELISA in xenograft models may enable more accurate measurement of the anti-cancer drug effects on cell proliferation and these results encourage further applications of TK 210 ELISA in drug development projects. Citation Format: Kiran Kumar Jagarlamudi, Ramesh Jayaraman, Sundaresh Babu, Martin Shaw, Staffan Eriksson. Evaluation of TK1 protein changes using the AroCell TK 210 ELISA in an acute myeloid leukemia (AML) xenograft model during 5-fluoruracil treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3130.

Abstract 379: Myeloperoxidase-produced HOCl is a paracrine effector linking myeloid cells to NF-κB signaling in melanoma, mediating anti-tumor responses during early melanoma progression

Abstract The myeloperoxidase (MPO) system of myeloid-derived cells (MDCs) is central to cellular innate immunity and the microbicidal activities of phagocytes, while also impacting the pathogenesis of many disorders, including cancer. Upon MDC activation, MPO is secreted into phagosomes, catalyzing the production of hypochlorous acid (HOCl). HOCl is a potent chlorinating oxidant with significant biological effects, but the mechanisms by which HOCl impacts cancer growth remain unresolved. Herein, we demonstrate that the myeloid lineage-restricted MPO-HOCl system has anti-tumor effects during early melanoma progression. Orthotopic melanomas grew slower in immuno-competent host mice wild type for MPO+/+ compared to syngeneic MPO-null (MPO-/-) animals. Using a specific and potent inhibitor of MPO, 4-aminobenzoic acid hydrazide (4-ABAH), melanoma tumor growth in MPO+/+ mice continuously administered 4-ABAH, pharmacologically mimicked the MPO-/- phenotype. Real-time intravital tumor imaging in vivo using skinfold window chamber animal models as well as live cell co-culture studies revealed a cell-cell proximity-dependent association between MDC-derived MPO activity and blockade of ligand-induced IκBα degradation in tumor cells. Real-time bioluminescent imaging and quantitative monitoring of the canonical NF-κB pathway was assessed in melanoma cells stably expressing bioluminescent reporters: a transcriptionally-activated NF-κB-promoter-driven Firefly luciferase (FLuc) reporter or a fusion reporter under the control of an NF-κB-responsive promoter fused to IκBα-FLuc reporter. HOCl was shown to directly inhibit IκB kinase (IKK) activity, thereby decreasing NF-κB transcriptional activation within melanomas, increasing circulating levels of the myeloid-attracting cytokines CXCL1 and CXCL5, enhancing local infiltration of CD8+ cytotoxic T cells, and dampening tumor growth. Overall, these data reveal a novel role for MDC-derived HOCl as a small molecule paracrine signaling factor that trans-inhibits IKK in melanoma cells, mediating anti-tumor responses during early tumor progression. Citation Format: Tracy W. Liu, Seth T. Gammon, Ping Yang, David T. Fuentes, David Piwnica-Worms. Myeloperoxidase-produced HOCl is a paracrine effector linking myeloid cells to NF-κB signaling in melanoma, mediating anti-tumor responses during early melanoma progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 379.

Abstract 1118: Absence of mouse-specific tumor evolution in patient-derived cancer xenografts

Abstract Patient-Derived Xenografts (PDXs) are preclinical models largely used to study tumor biology and drug response. Recent literature highlighted the possibility that growth of human tumors in a mouse microenvironment imposes a selection driving mouse-specific genetic evolution of PDXs, which may compromise their reliability as human cancer models. Conversely, independent studies observed a conservation of the genomic landscape during PDX engraftment and passaging. We noticed that PDX genetic evolution was particularly evident in studies based on copy number aberration (CNA) inferred from gene expression data, while it was negligible when DNA-based CNA profiles were employed. Therefore, in a joint international effort of the EurOPDX and PDXNet consortia, we assembled a dataset of 37 hepatocellular and 54 gastric carcinoma tumor or PDX samples with matched RNA-based and DNA-based CNA profiles. We found that DNA-based CNA profiles invariably yield higher concordance between patient's tumor and derived PDXs than those inferred from RNA. RNA-based profiles displayed poor concordance with matched DNA-based profiles, and much lower resolution, so that they missed many focal copy number events detected by DNA-based methods. These results revealed that CNA measurements cannot be accurately estimated by expression data and that a systematic reassessment of CNA dynamics in PDXs based on DNA data is required. To this aim, we generated CNA profiles by low-pass whole genome sequencing (WGS) of 87 colorectal and 43 breast cancer triplets, each composed of matched patient's tumor (PT) and PDX at early (PDX-early) and later (PDX-late) passage. In this way, for each tumor type, we generated three perfectly matched PT, PDX-early and PDX-late cohorts and performed CNA recurrence analysis by GISTIC in each cohort. The hypothesis was that if the mouse host induces a selective pressure capable of shaping the CNA landscape during PDX engraftment and propagation, GISTIC analysis would highlight systematic and progressive changes, from the PT to the PDX-early cohort, and then to the PDX-late cohort. Notably instead, the CNA profiles of the PT and PDX-early/late cohorts were virtually indistinguishable, with no progressive accumulation or loss of CNA during PDX passage and only minor changes not functionally related or associated to cancer-driver or actionable genes. These results were not consequence of insufficient capture of the CNA repertoire, since the GISTIC profiles recapitulated those generated by TCGA for colorectal and breast cancer. In summary, our analyses highlighted that while RNA-based CNA inferences have inadequate resolution and accuracy to study genomic evolution in PDXs, DNA-based CNA profiles confirm retention of CNAs in PTs and PDXs, excluding a systematic mouse driven selection via copy number changes. Ultimately, these results support the robustness of PDXs as preclinical models for predicting drug response. Citation Format: Jessica Giordano, Xing Yi Woo, Anuj Srivastava, Zi-Ming Zhao, Michael W. Lloyd, Roebi de Bruijn, Yun-Suhk Suh, Francesco Galimi, Andrea Bertotti, Adam Lafferty, Alice C. O'Farrell, Elodie Modave, Diether Lambrechts, Petra ter Brugge, Violeta Serra, Elisabetta Marangoni, Rania El Botty, Jong-Il Kim, Han-Kwang Yang, Charles Lee, Dennis A. Dean, Brandi Davis-Dusenbery, Yvonne A. Evrard, James H. Doroshow, Alana L. Welm, Bryan E. Welm, Michael T. Lewis, Bingliang Fang, Jack Roth, Funda Meric-Bernstam, Meenhard Herlyn, Michael Davies, Li Ding, Shunqiang Li, Ramaswamy Govindan, Jeffrey A. Moscow, Carol J. Bult, Claudio Isella, Livio Trusolino, Annette T. Byrne, Jos Jonkers, Jeffrey H. Chuang, Enzo Medico, EurOPDX consortium & PDXNET consortium. Absence of mouse-specific tumor evolution in patient-derived cancer xenografts [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1118.

Abstract 1680: Assessment of statistical power in one mouse per treatment design for preclinical anticancer agent PDX large scale drug screens

Abstract Background: Patient-derived tumor xenograft (PDX) models are increasingly used to evaluate the effectiveness of preclinical anticancer agents. To test many anticancer agents simultaneously a large scale drug screen can be utilized with a one mouse per treatment (1 × 1) design. With this approach, typically only treatments found to be effective are used in further studies. We investigated the rates of “false negatives” where drugs are being incorrectly found ineffective in initial screens and potentially not considered again. We focused on modifiable parameters which could increase the statistical power (rate of true positives) of this design based on recent PDX lung experiments. Methods: We used PDX drug screen studies from our lab as a reference for tumor growth rate and mouse variation. Studies included 43 non-small cell lung cancer PDX experiments testing a total of 14 different anti-cancer agents. Each experiment included on average 6 replicates per group (531 total mice), from 25 unique PDX models. In each experiment PDX models were established from patient tumor fragments that were implanted at the flank of immunodeficient mouse hosts. Xenograft tumor fragments were expanded into mouse replicates to test with anti-cancer agents. The standard protocol was treatment with agents at doses with reported in vivo antitumor effects. Tumor size was measured twice weekly. This presented us a distribution of treatment effect sizes, and across mouse variation. We assessed the statistical power of the 1 × 1 design under different settings to determine if/when the design would be appropriate. Settings included; modifying the treatment effect size, mouse variation, and follow up schedule. The estimated treatment effect sizes were divided at the tertiles which we refer to as small, medium, and large. Mouse variation was assessed at the median value (average variation) and at the first quartile (small variation). We assumed a typical measurement schedule to be twice a week for four weeks, and a more intense schedule as three times a week. We used a relaxed 0.2 alpha level when calculating the power rates. Results: Treatments with a large effect have a 98% statistical power under the assumption of average variance and typical measurement schedule. For medium and small treatment effects the statistical power are 67% and 41% respectively. A more intense measurement schedule and small variation increases the statistical power to 99%, 70% and 43%, depending on the effect size. Conclusion: In contrast to large effect sizes which can be detected easily under a 1 × 1 design, the medium and small effect sizes have a large chance of being rejected. A treatment with a median effect under optimal circumstance will have a 30% change of being rejected, and a small treatment effect will have a 57% chance of being rejected. In conclusion a 1 × 1 design is appropriate only when there is a belief the that treatment is very effective. Citation Format: Jessica Weiss, Nhu-An Pham, Melania Pintilie, Ming Li, Ming Tsao, Geoffrey Liu, Wei Xu. Assessment of statistical power in one mouse per treatment design for preclinical anticancer agent PDX large scale drug screens [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1680.

Long-Term Monitoring of Donor Xenogeneic Testis Tissue Grafts and Cell Implants in Recipient Mice Using Ultrasound Biomicroscopy

Testis tissue xenografting and testis cell aggregate implantation from various donor species into recipient mice are novel models for the study and manipulation of testis formation and function in target species. Thus far, the analysis of such studies has been limited to surgical or post-mortem retrieval of samples. Here we used ultrasound biomicroscopy (UBM) to monitor the development of neonatal porcine testis grafts and implants in host mice for 24 wk, and to correlate UBM and (immuno)histologic changes. This led to long-term visualization of gradual changes in volume, dimension and structure of grafts and implants; detection of a 4 wk developmental gap between grafts and implants; and revelation of differences in implant development depending on the craniocaudal site of implantation on the back of host mice. Our data support the reliability and precision of UBM for longitudinal study of transplants, which eliminates the need for frequent surgical sampling.

Effect of Dinotefuran, Permethrin, and Pyriproxyfen (Vectra® 3D) on the Foraging and Blood-Feeding Behaviors of Aedes albopictus Using Laboratory Rodent Model.

Simple SummaryTiger mosquito (Aedes albopictus) is a harmful vector involved in the transmission of several diseases to humans and their pets. Currently, several veterinary products are used to prevent pets against bites of arthropod vectors. However, there is no available information on the effect of these products on feeding and host choice behaviors of Aedes albopictus in the presence of treated and untreated hosts, as is the case of treated dogs present in close physical contact with their owners. The present study investigated the effect of a spot-on product (Vectra® 3D) on the feeding and host choice behaviours of Aedes albopictus when treated and untreated hosts are presents. Laboratory rodent model was performed to simulate the natural conditions. Rat and mouse hosts were alternately treated with Vectra® 3D and exposed simultaneously to starved mosquitoes. Results showed that Vectra® 3D-treated hosts are perfectly protected against up to 82% of mosquitoes. While up to 21% of mosquitoes were repelled from untreated hosts when these latter are present in close physical contact (30 cm) with treated ones suggesting an indirect protection that can allowed the protection of owners who treat their pets with Vectra® 3D.Dinotefuran-Permethrin-Pyriproxyfen (DPP) is used to kill and repel mosquitoes from dogs. However, the influence of the product on the host-seeking behavior of mosquitoes remains unknown. The interference of DPP with the host selection of unfed female Aedes albopictus was investigated. A total of 18 animals (9 mice and 9 rats) were divided into three groups of six animals each. DU: DPP treated rats (n = 3) with untreated mice (n = 3), UD: DPP treated mice (n = 3) with untreated rats (n = 3) and control UU: untreated mice (n = 3) and untreated rats (n = 3). In each group, the rats and mice were placed 30 cm apart. After sedation, the animals in each group were exposed twice (Day 1 and Day 7 post-treatment) for one hour to 71 ± 3 female mosquitoes. Mosquitoes were categorized after the 2-h post-exposure period as dead or alive. Blood-meal origin was determined from mosquitoes using a newly customized duplex qPCR. The highest values of forage ratio (1.36 ≥ wi ≤ 1.88) and selection index (0.63 ≥ Bi ≤ 0.94) for rat hosts indicates a preference of mosquitoes for this species as compared to mice when co-housed during the exposure. The mosquitoes only seldom fed on mice, even in the untreated group. The anti-feeding effect of DPP was therefore only assessed on rat’s hosts. The results showed that DPP, when directly applied on rats, provided a direct protection of 82% and 61% on Day 1 and Day 7, respectively, while when applied on mice hosts (UD), the DPP provided an indirect protection of 21% and 10% on Day 1 and Day 7, respectively. The results showed also that DPP, when applied on rats, provided a direct protection against Ae. albopictus bites. This effect did not result in increased exposure of the untreated host placed in the same cage at a distance of 30 cm.

Baylisascaris Potosis Larvae in Mice of Different Strains and Infectivity of Tissue Larvae.

SummaryMigration of Baylisascaris potosis larvae in different mouse strains were compared, and infectivity of the persisting larvae in mice tissues were investigated. Five strains of mice, BALB/c, C57BL/6, AKR, B10.BR, and ICR were inoculated with 1,000 B. potosis eggs/mouse, and necropsied at week 13 post inoculation (PI). The other uninfected ICR mice (secondary host) were inoculated with 43 larvae/ mouse recovered from mice at week 13 PI with eggs, and necropsied at day 21 PI. Larvae in organs or tissues were counted at necropsy. One AKR mouse showed torticollis and circling at day 56 PI. At necropsy at week 13 PI, larvae were recovered from all mice. A mean total larvae recovered were 124.1 (n=40). Majority of larvae were found in the carcass (mean 113.9) and some in the viscera (mean 9.9). Zero to 1 larva were found in the brain or eyes of some mice. There were no differences among the mouse strains in the number of larvae, except in the viscera; more larvae were seen in BALB/c or ICR than in B10.BR mice. No larvae were found in the secondary host mice. Present study demonstrated that B. potosis larvae migrate well in the carcass of any strains of mice, however, the tissue larvae did not infect the secondary host. Results of our present study suggest that B. potosis larvae is less aggressive for the nervous tissue migration than that of B. procyonis larvae which is commonly known to migrate in central nervous system of mammals and birds.

Intracisternal injection of beta-amyloid seeds promotes cerebral amyloid angiopathy

Beta amyloid (Aβ) is a key component of parenchymal Aβ plaques and vascular Aβ fibrils, which lead to cerebral amyloid angiopathy (CAA) in Alzheimer’s disease (AD). Recent studies have revealed that Aβ contained in the cerebrospinal fluid (CSF) can re-enter into brain through paravascular spaces. However, whether Aβ in CSF may act as a constant source of pathogenic Aβ in AD is still unclear. This study aimed to examine whether Aβ pathology could be worsened when CSF Aβ level was enhanced by intra-cisternal infusion of aged brain extract containing abundant Aβ in TgCRND8 host mice. TgCRND8 mouse is an AD animal model which develops predominant parenchymal Aβ plaques in the brain at as early as 3 months of age. Here, we showed that single intracisternal injection of Aβ seeds into TgCRND8 mice before the presence of Aβ pathology induced robust prion-like propagation of CAA within 90 days. The induced CAA is mainly distributed in the cerebral cortex, hippocampus and thalamus of TgCRND8 mice. Surprisingly, despite the robust increase in CAA levels, the TgCRND8 mice had a marked decrease in parenchymal Aβ plaques and the plaques related neuroinflammation in the brains compared with the control mice. These results amply indicate that Aβ in CSF may act as a source of Aβ contributing to the growth of vascular Aβ deposits in CAA. Our findings provide experimental evidence to unravel the mechanisms of CAA formation and the potential of targeting CSF Aβ for CAA.

A comparison of prostatic development in xenografts of human fetal prostate and human female fetal proximal urethra grown in dihydrotestosterone-treated hosts

The goal of this paper is to explore the ability of the human female urogenital sinus immediately below the bladder (proximal urethra) to undergo prostatic development in response to dihydrotestosterone (DHT). To establish this idea, xenografts of human fetal female proximal urethra were grown in castrated nude mouse hosts receiving a subcutaneous DHT pellet. To verify the prostatic nature of the resultant glands, DHT-treated human fetal female urethral xenografts were compared with human fetal prostatic xenografts (derived from male specimens) grown in untreated and DHT-treated castrated mouse hosts and human fetal female proximal urethral xenografts grown in untreated castrated hosts. The resultant glands observed in DHT-treated human fetal female proximal urethral xenografts expressed 3 prostate-specific markers, NKX3.1, prostate specific antigen and prostatic acid phosphatase as well as the androgen receptor. Glands induced by DHT exhibited a protein expression profile of additional immunohistochemical markers (seven keratins, RUNX1, ESR2, TP63 and FOXA1) consistent with the unique spatial pattern of these proteins in prostatic ducts. Xenografts of human fetal female proximal urethra grown in DHT-treated hosts also expressed one of the salient features of prostatic development, namely androgen responsiveness. The experimental induction of prostatic differentiation from human fetal female proximal urethra makes possible future in-depth analysis of the molecular pathways directly involved in initiation of human prostatic development and subsequent epithelial differentiation, and more important whether the molecular pathways involved in human prostatic development are similar/identical versus different from that in murine prostatic development.

Extracellular vesicles from Heligmosomoides bakeri and Trichuris muris contain distinct microRNA families and small RNAs that could underpin different functions in the host

Extracellular vesicles (EVs) have emerged as a ubiquitous component of helminth excretory-secretory products that can deliver parasite molecules to host cells to elicit immunomodulatory effects. RNAs are one type of cargo molecule that can underpin EV functions, hence there is extensive interest in characterising the RNAs that are present in EVs from different helminth species. Here we outline methods for identifying all of the small RNAs (sRNA) in helminth EVs and address how different methodologies may influence the sRNAs detected. We show that different EV purification methods introduce relatively little variation in the sRNAs that are detected, and that different RNA library preparation methods yielded larger differences. We compared the EV sRNAs in the gastrointestinal nematode Heligmosomoides bakeri with those in EVs from the distantly related gastrointestinal nematode Trichuris muris, and found that many of the sRNAs in both organisms derive from repetitive elements or intergenic regions. However, only in H. bakeri do these RNAs contain a 5′ triphosphate, and Guanine (G) starting nucleotide, consistent with their biogenesis by RNA-dependent RNA polymerases (RdRPs). Distinct microRNA (miRNA) families are carried in EVs from each parasite, with H. bakeri EVs specific for miR-71, miR-49, miR-63, miR-259 and miR-240 gene families, and T. muris EVs specific for miR-1, miR-1822 and miR-252, and enriched for miR-59, miR-72 and miR-44 families, with the miR-9, miR-10, miR-80 and let-7 families abundant in both. We found a larger proportion of miRNA reads derive from the mouse host in T. muris EVs, compared with H. bakeri EVs. Our report underscores potential biases in the sRNAs sequenced based on library preparation methods, suggests specific nematode lineages have evolved distinct sRNA synthesis/export pathways, and highlights specific differences in EV miRNAs from H. bakeri and T. muris that may underpin functional adaptation to their host niches.

Irradiation dependent inflammatory response may enhance satellite cell engraftment

Skeletal muscle stem (satellite) cells transplanted into host mouse muscles contribute to muscle regeneration. Irradiation of host muscle enhances donor stem cell engraftment by promoting the proliferation of transplanted donor cells. We hypothesised that, similar to other systems, cells damaged by radiation might be effecting this donor cell proliferation. But we found no difference in the percentage of dying (TUNEL+) cells in immunodeficient dystrophic mouse muscles at the times after the irradiation dose that enhances donor cell engraftment. Similarly, irradiation did not significantly increase the number of TUNEL+ cells in non-dystrophic immunodeficient mouse muscles and it only slightly enhanced donor satellite cell engraftment in this mouse strain, suggesting either that the effector cells are present in greater numbers within dystrophic muscle, or that an innate immune response is required for effective donor cell engraftment. Donor cell engraftment within non-irradiated dystrophic host mouse muscles was not enhanced if they were transplanted with either satellite cells, or myofibres, derived from irradiated dystrophic mouse muscle. But a mixture of cells from irradiated muscle transplanted with donor satellite cells promoted donor cell engraftment in a few instances, suggesting that a rare, yet to be identified, cell type within irradiated dystrophic muscle enhances the donor stem cell-mediated regeneration. The mechanism by which cells within irradiated host muscle promote donor cell engraftment remains elusive.

Abstract A06: Impact of cigarette smoke exposure on acute myeloid leukemia progression

Abstract Cigarette smoke is known to cause lung cancer but is also associated with increased incidence of leukemia. Acute myeloid leukemia (AML) is the most lethal form of leukemia, and patients who are or were smokers have increased risk of developing AML and have worse prognosis. There is also an increased incidence of AML in children whose parents smoked either during or after the pregnancy. It is currently unknown how cigarette smoke exposure (CSE) may be impacting leukemic progression or treatment efficacy in AML. Cigarette smoking increases levels of reactive oxygen species (ROS) in the tissue of smokers, including peripheral blood samples. Oxidative stress can alter epigenetic signaling, including DNA methylation. Oxidation of methylated DNA and oxidative lesions on DNA can alter normal DNA methylation patterns. Smokers have altered DNA methylation that can be retained decades after the cessation of the habit, and these changes can be found in the blood cells of smokers. Cigarette smoke condensate (CSC) treatment has also been seen to epigenetically prime nontransformed human bronchial cells for malignant transformation. These occurrences have been seen in the context of lung cancer and lung damage, but studies into other tissue damage have largely been overlooked. Thus, we hypothesize that CSE impacts leukemic progression and treatment in AML through these mechanisms. We have developed a model to study the impact of CSE on AML in mice. NOD-SCID mice are smoke exposed for two weeks prior to being engrafted with luciferase-labeled human AML cells. The CSE is performed in the SCIREQ Cigarette Smoking Robot, with 3R4F research cigarettes 5 days/week. We include experimental arms of smoking cessation (who stop once evidence of leukemic engraftment is detected) and continued smoking, to model patients who either stop or continue smoking upon diagnosis of AML. Leukemic burden is quantified through bioluminescent imaging throughout the experiment. From these models, we have observed that two weeks of CSE is sufficient to significantly increase (p-value<0.0001) early leukemic burden as compared to non-smoke exposed mice, in FLT3-ITD AML-bearing mice. Spleen samples revealed trends of increased levels of superoxides in the CSE mice. Currently, immunohistochemistry is being performed on collected organs to detect oxidative stress markers. Additionally, mouse spleen DNA underwent reduced representative bisulfide sequencing; the levels of DNA methylation in the mouse host cells and in the leukemia are compared between our CSE groups. This is the first description of CSE promoting leukemia growth in mouse model, which will enable the study of mechanistic aspects. This may help define if unique treatment strategies are needed for these patients. Citation Format: Yue Lu, Marcos Estecio, Marina Konopleva, Elias Jabbour, Joya Chandra, Mary Figueroa. Impact of cigarette smoke exposure on acute myeloid leukemia progression [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A06.