Passage In Mice Research Articles

Mutations in nonstructural proteins essential for pathogenicity in SARS-CoV-2-infected mice.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has resulted in substantial morbidity and mortality. The basis of severe disease in humans is difficult to determine without the use of experimental animal models. Mice are resistant to infection with ancestral strains of SARS-CoV-2, although many variants that arose later in the pandemic were able to directly infect mice. In almost all cases, viruses that naturally infected mice or were engineered to enable mouse infection required mouse passage to become virulent. In most cases, changes in structural and nonstructural changes occurred during mouse adaptation. However, the mechanism of increased virulence in mice is not understood. Here, using a recently described strain of mouse-adapted SARS-CoV-2 (rSARS2-MA30N501Y), we engineered a series of recombinant viruses that expressed a subset of the mutations present in rSARS2-MA30N501Y. Mutations were detected in the spike protein and in three nonstructural proteins (nsp4, nsp8, and nsp9). We found that infection of mice with recombinant SARS-CoV-2 expressing only the S protein mutations caused very mild infection. Addition of the mutations in nsp4 and nsp8 was required for complete virulence. Of note, all these recombinant viruses replicated equivalently in cultured cells. The innate immune response was delayed after infection with virulent compared to attenuated viruses. Further, using a lineage tracking system, we found that attenuated virus was highly inhibited in the ability to infect the parenchyma, but not the airway after infection. Together, these results indicate that mutations in both the S protein and nonstructural proteins are required for maximal virulence during mouse adaptation.IMPORTANCEUnderstanding the pathogenesis of coronavirus disease 2019 (COVID-19) requires the study of experimental animals after infection with severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). For this purpose, several mouse-adapted SARS-CoV-2 strains have been developed. Here, using a strain of mouse-adapted virus that causes a range of diseases ranging from mild to severe, we show that mutations in both a structural protein [spike (S) protein] and nonstructural proteins are required for maximal virulence. Thus, changes in the S protein, the most widely studied viral protein, while required for mouse adaptation, are not sufficient to result in a virulent virus.

Humane Endpoints in Swiss Webster Mice Infected with Toxoplasma gondii RH Strain.

The highly virulent Toxoplasma gondii RH strain is maintained through successive passages in mice, but there is still a lack of studies that refine these procedures from a 3Rs perspective, where humanitarian ideals aim to minimize the stress, pain, or suffering of the animals used in the research without the loss of results. The aim of this study was to establish humane endpoints in Swiss Webster mice inoculated with the T. gondii RH strain. A total of 52 mice were infected with 5 × 106 tachyzoites/mL and monitored for periods of up to 5 days. The parameters body weight; hair condition; higher than normal body temperature; hypothermia; respiratory function; pain; soft stools or diarrhea; bloody diarrhea; tense, nervous, or in distress during handling; and ascites were recorded daily in score tables. The results showed that prominent piloerection, respiratory function, pain parameters, and ascites are important clinical signs to be used as a cut-off point for implementing euthanasia. The application of this refinement method helped to avoid animal suffering and pain without compromising the number of parasites recovered. We therefore suggest adopting these parameters in research protocols that require the maintenance of the T. gondii RH strain in murine models to avoid and reduce animal suffering.

Abstract 536: Breaking through ADCs resistance: Constructing preclinical enhertu-induced resistant model

Abstract Antibody-drug conjugates (ADCs) are compounds that combine the specificity of monoclonal antibodies (mAbs) with the cytotoxic payload of toxic substances. In recent years, ADCs have rapidly developed in the field of cancer therapy, particularly in the treatment of HER2-positive breast cancer. One representative example is Trastuzumab deruxtecan (Enhertu), which has become a new benchmark for anti-HER2 ADCs due to its excellent performance in clinical trials. Despite the impressive clinical outcomes of ADCs, some tumors in patients may still develop resistance to these drugs. Therefore, it is crucial to understand the mechanisms of resistance and develop strategies to overcome ADCs resistance. To achieve these goals, it is essential to establish and utilize preclinical models to study these resistance mechanisms. Preclinical models of resistance to ADCs have mainly been generated using human cultured cell lines. JIMT-1 is a HER2-positive breast cancer cell line that exhibits primary resistance to the anti-HER2 antibody trastuzumab and also shows resistance to trastuzumab-derived ADC T-DM1. However, we found that Enhertu significantly inhibited tumor growth in JIMT-1 xenograft models. We subjected JIMT-1 tumor tissues to continuous passage in mice and administered long-term drug induction. The tumor growth rate gradually increased, and the drug sensitivity decreased, resulting in the acquisition of an Enhertu-resistant tumor model. The obtained resistant tumor tissues not only include tumor cells themselves but also comprise fibroblasts, extracellular matrix, and other cellular components, enabling a more comprehensive investigation into the interactions between different cells within the tumor microenvironment (TME). We believe that the establishment of preclinical Enhertu-resistant models helps predict resistance that has not yet emerged clinically and provides references for future drug development. Citation Format: Yuan Fang, Huiyi Wang, Jun Xing, Lu Yang, Jing Zhao, Xiang Gao, Cunxiang Ju. Breaking through ADCs resistance: Constructing preclinical enhertu-induced resistant model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 536.

Restoration of virulence in the attenuated Candid#1 vaccine virus requires reversion at both positions 168 and 427 in the envelope glycoprotein GPC.

Live-attenuated virus vaccines provide long-lived protection against viral disease but carry inherent risks of residual pathogenicity and genetic reversion. The live-attenuated Candid#1 vaccine was developed to protect Argentines against lethal infection by the Argentine hemorrhagic fever arenavirus, Junín virus. Despite its safety and efficacy in Phase III clinical study, the vaccine is not licensed in the US, in part due to concerns regarding the genetic stability of attenuation. Previous studies had identified a single F427I mutation in the transmembrane domain of the Candid#1 envelope glycoprotein GPC as the key determinant of attenuation, as well as the propensity of this mutation to revert upon passage in cell culture and neonatal mice. To ascertain the consequences of this reversion event, we introduced the I427F mutation into recombinant Candid#1 (I427F rCan) and investigated the effects in two validated small-animal models: in mice expressing the essential virus receptor (human transferrin receptor 1; huTfR1) and in the conventional guinea pig model. We report that I427F rCan displays only modest virulence in huTfR1 mice and appears attenuated in guinea pigs. Reversion at another attenuating locus in Candid#1 GPC (T168A) was also examined, and a similar pattern was observed. By contrast, virus bearing both revertant mutations (A168T+I427F rCan) approached the lethal virulence of the pathogenic Romero strain in huTfR1 mice. Virulence was less extreme in guinea pigs. Our findings suggest that genetic stabilization at both positions is required to minimize the likelihood of reversion to virulence in a second-generation Candid#1 vaccine.IMPORTANCELive-attenuated virus vaccines, such as measles/mumps/rubella and oral poliovirus, provide robust protection against disease but carry with them the risk of genetic reversion to the virulent form. Here, we analyze the genetics of reversion in the live-attenuated Candid#1 vaccine that is used to protect against Argentine hemorrhagic fever, an often-lethal disease caused by the Junín arenavirus. In two validated small-animal models, we find that restoration of virulence in recombinant Candid#1 viruses requires back-mutation at two positions specific to the Candid#1 envelope glycoprotein GPC, at positions 168 and 427. Viruses bearing only a single change showed only modest virulence. We discuss strategies to genetically harden Candid#1 GPC against these two reversion events in order to develop a safer second-generation Candid#1 vaccine virus.

Abstract B015: A novel Cluster 3 Endometrial Cancer PDX model to test hormone therapy regimens

Abstract Endometrial cancer is the most commonly diagnosed gynecologic cancer and continues to rise in incidence and mortality rates worldwide. With the evaluation of large datasets including the TCGA, endometrial cancers have been subdivided into four different cluster groups based on molecular characteristics. The most common subtype of endometrial cancer is the Cluster 3 category, demarcated by the expression of estrogen receptors (ER) and progesterone receptors (PR) with the potential to respond to hormonal therapy. However, Cluster 3 tumors frequently lose hormone receptor expression over time, particularly PR, making them resistant to treatment. Although numerous cell line models exist to study this disease, there is a need for improved models to select best treatment options for the specific types of endometrial cancers. Here, we describe the development of an endometrial cancer patient derived xenograft (PDX), EC-PDX3. The initial tumor from which EC-PDX3 was derived was a low-grade endometrioid endometrial carcinoma that contained a low number of somatic copy number alterations. The tumor initially tested robustly positive for both ER and PR in the donor patient as well as in initial passages in athymic mice, making this model valuable for hormone therapy research. In these studies, EC-PDX3 cells were grown within immunodeficient NSG mice. We performed H&E, ER and PR staining prior to experimentation and found that while ER remained highly positive, PR levels were significantly downregulated, indicating the development of a hormone resistant state similar to that occurring in patients over time. To determine if these cells respond to hormonal therapy and if PR expression could be induced, mice were initially treated with estradiol to enhance PR expression. Next, mice were further treated with progesterone or onapristone, an experimental progestin agonist/antagonist with previously reported encouraging activity in advanced endometrial cancer in phase I and II trials. Tumors were measured weekly to determine hormone effects on cancer growth. Mice treated with estradiol alone showed substantially larger tumor growth as compared to the estradiol + progesterone and estradiol + onapristone treatment groups, indicating that hormone sensitivity was likely re-instated. RNA sequencing using Ishikawa cells in culture revealed the transcriptional effects of estradiol, progesterone and onapristone which complement the data obtained from EC-PDX3 bearing mice treated similarly. These studies identify interesting growth inhibitory effects of onapristone in endometrial cancer cells primed with estradiol including the significant induction of the cell cycle regulators such as p21 and genes involved in apoptosis. In addition, EC-PDX3 provides a model to assess hormonal therapy in Cluster 3 tumors that initially respond but develop resistance to treatment over time. Citation Format: Kimberly K. Leslie, Lane E. Smith, Jamie Padilla, Adrian Quintana. A novel Cluster 3 Endometrial Cancer PDX model to test hormone therapy regimens [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B015.

Wild Bird-Origin H6N2 Influenza Virus Acquires Enhanced Pathogenicity after Single Passage in Mice.

The H6 subtype of avian influenza viruses (AIVs) has emerged as one of the predominant subtypes in both wild and domestic avian species. Currently, H6 AIVs have acquired the ability to infect a wide range of mammals, though the related molecular mechanisms have yet to be fully investigated. In this study, a wild bird-origin H6N2 AIV was isolated from the East Asian-Australasian migratory flyway region located in Liaoning Province. This H6N2 virus initially expressed limited replication in mice. However, after one passage in mice, the virus acquired two mutations, PB2 E627K and HA A110V. The mutant displayed enhanced replication both in vitro and in vivo, proving lethal to mice. But the mutant retained the α-2, 3-linked sialic acid binding property and failed to transmit in guinea pigs. We explored the molecular mechanisms underlying the pathogenicity difference between the wild type and the mutant. Our findings revealed that PB2 E627K dramatically enhanced the polymerase activity of the H6N2 virus, while the HA A110V mutation decreased the pH of HA activation. This study demonstrated that the H6N2 subtype wild bird-origin AIV easily acquired the mammalian adaptation. The monitoring and evaluation of H6 wild bird-origin AIV should be strengthened.

ELK1/MTOR/S6K1 Pathway Contributes to Acquired Resistance to Gefitinib in Non-Small Cell Lung Cancer.

The development of acquired resistance to small molecule tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR) signaling has hindered their efficacy in treating non-small cell lung cancer (NSCLC) patients. Our previous study showed that constitutive activation of the 70 kDa ribosomal protein S6 kinase 1 (S6K1) contributes to the acquired resistance to EGFR-TKIs in NSCLC cell lines and xenograft tumors in nude mice. However, the regulatory mechanisms underlying S6K1 constitutive activation in TKI-resistant cancer cells have not yet been explored. In this study, we recapitulated this finding by taking advantage of a gefitinib-resistant patient-derived xenograft (PDX) model established through a number of passages in mice treated with increasing doses of gefitinib. The dissociated primary cells from the resistant PDX tumors (PDX-R) displayed higher levels of phosphor-S6K1 expression and were resistant to gefitinib compared to cells from passage-matched parental PDX tumors (PDX-P). Both genetic and pharmacological inhibition of S6K1 increased sensitivity to gefitinib in PDX-R cells. In addition, both total and phosphorylated mechanistic target of rapamycin kinase (MTOR) levels were upregulated in PDX-R and gefitinib-resistant PC9G cells. Knockdown of MTOR by siRNA decreased the expression levels of total and phosphor-S6K1 and increased sensitivity to gefitinib in PDX-R and PC9G cells. Moreover, a transcription factor ELK1, which has multiple predicted binding sites on the MTOR promoter, was also upregulated in PDX-R and PC9G cells, while the knockdown of ELK1 led to decreased expression of MTOR and S6K1. The chromatin immunoprecipitation (ChIP)-PCR assay showed the direct binding between ELK1 and the MTOR promoter, and the luciferase reporter assay further indicated that ELK1 could upregulate MTOR expression through tuning up its transcription. Silencing ELK1 via siRNA transfection improved the efficacy of gefitinib in PDX-R and PC9G cells. These results support the notion that activation of ELK1/MTOR/S6K1 signaling contributes to acquired resistance to gefitinib in NSCLC. The findings in this study shed new light on the mechanism for acquired EGFR-TKI resistance and provide potential novel strategies by targeting the ELK1/MTOR/S6K1 pathway.

In vivo evolution of antimicrobial resistance in a biofilm model of Pseudomonas aeruginosa lung infection.

The evolution of antimicrobial resistance (AMR) in biofilms has been repeatedly studied by experimental evolution in vitro, but rarely in vivo. The complex microenvironment at the infection site imposes selective pressures on the bacterial biofilms, potentially influencing the development of AMR. We report here the development of AMR in an in vivo mouse model of Pseudomonas aeruginosa biofilm lung infection. The P. aeruginosa embedded in seaweed alginate beads underwent four successive lung infection passages with or without ciprofloxacin (CIP) exposure. The development of CIP resistance was assessed at each passage by population analysis of the bacterial populations recovered from the lungs of CIP-treated and control mice, with subsequent whole-genome sequencing of selected isolates. As inflammation plays a crucial role in shaping the microenvironment at the infection site, its impact was explored through the measurement of cytokine levels in the lung homogenate. A rapid development of AMR was observed starting from the second passage in the CIP-treated mice. Genetic analysis revealed mutations in nfxB, efflux pumps (mexZ), and two-component systems (parS) contribution to CIP resistance. The control group isolates exhibited mutations in the dipA gene, likely associated with biofilm dispersion. In the initial two passages, the CIP-treated group exhibited an elevated inflammatory response compared to the control group. This increase may potentially contribute to the release of mutagenic reactive oxygen species and the development of AMR. In conclusion, this study illustrates the complex relationship between infection, antibiotic treatment, and immune response.

The adaptive potential of North American subtype H7N2 avian influenza viruses to mammals

Introduction. H7 subtype avian influenza viruses causing severe epizootics among birds are phylogenetically different in the Eastern and Western hemispheres. Numerous human infections caused by these viruses in the Eastern hemisphere indicate that H7 viruses can overcome the interspecies barrier and pose a potential threat of a new pandemic.The H7N2 viruses with deletion of amino acids 221–228 (H3 numbering) in hemagglutinin (HA) had been circulating among poultry in the Western Hemisphere during 1996–2006, and had once again been detected in 2016 in an animal shelter, where they caused cat diseases.
 The objective of this study is to elucidate the mechanism of adaptation to mammals of North American H7N2 influenza viruses with deletion in HA.
 Materials and methods. The A/chicken/New Jersey/294598-12/2004 (H7N2) virus was adapted to mice by the lung passages. Complete genomes of original and mouse-adapted viruses were analyzed. The receptor specificity and thermostability of viruses, HA activation pH and virulence for mice were determined.
 Results. The non-pathogenic H7N2 avian influenza virus became pathogenic after 10 passages in mice. Amino acid substitutions occurred in five viral proteins: one in PB2 (E627K), NA (K127N), NEP (E14Q), four in HA and six in NS1. Mutations in HA slightly changed receptor specificity but increased the pH of HA activation by 0.4 units. The NS1 protein undergone the greatest changes in the positions (N73T, S114G, K118R, G171A, F214L and G224R), where amino acid polymorphisms were observed in the original virus, but only minor amino acid variants have been preserved in the mouse adapted variant.
 Conclusion. The results show that H7N2 viruses have the potential to adapt to mammals. The increase in virulence is most likely due to the adaptive E627K mutation in PB2 and possibly in HA.

Exploring the alternative virulence determinants PB2 S155N and PA S49Y/D347G that promote mammalian adaptation of the H9N2 avian influenza virus in mice

The occurrence of human infections caused by avian H9N2 influenza viruses has raised concerns regarding the potential for human epidemics and pandemics. The molecular basis of viral adaptation to a new host needs to be further studied. Here, the bases of nucleotides 627 and 701 of PB2 were changed according to the uncoverable purine-to-pyrimidine transversion to block the development of PB2 627K and 701N mutations during serial passaging in mice. The purpose of this experiment was to identify key adaptive mutations in polymerase and NP genes that were obscured by the widely known host range determinants PB2 627K and 701N. Mouse-adapted H9N2 variants were obtained via twelve serial lung-to-lung passages. Sequence analysis showed that the mouse-adapted viruses acquired several mutations within the seven gene segments (PB2, PB1, PA, NP, HA, NA, and NS). One variant isolate with the highest polymerase activity possessed three substitutions, PB2 S155N, PA S49Y and D347G, which contributed to the highly virulent and mouse-adaptative phenotype. Further studies demonstrated that these three mutations resulted in increased polymerase activity, viral transcription and replication in mammalian cells, severe interstitial pneumonia, excessive inflammatory cellular infiltration and increased growth rates in mice. Our results suggest that the substitution of these three amino acid mutations may be an alternative strategy for H9N2 avian influenza viruses to adapt to mammalian hosts. The continued surveillance of zoonotic H9N2 influenza viruses should also include these mammalian adaptation markers as part of our pandemic preparedness efforts.

Comprehensive characterization of patient-derived xenograft models of pediatric leukemia

Patient-derived xenografts (PDX) remain valuable models for understanding the biology and for developing novel therapeutics. To expand current PDX models of childhood leukemia, we have developed new PDX models from Hispanic patients, a subgroup with a poorer overall outcome. Of 117 primary leukemia samples obtained, successful engraftment and serial passage in mice were achieved in 82 samples (70%). Hispanic patient samples engrafted at a rate (51/73, 70%) that was similar to non-Hispanic patient samples (31/45, 70%). With a new algorithm to remove mouse contamination in multi-omics datasets including methylation data, we found PDX models faithfully reflected somatic mutations, copy-number alterations, RNA expression, gene fusions, whole-genome methylation patterns, and immunophenotypes found in primary tumor (PT) samples in the first 50 reported here. This cohort of characterized PDX childhood leukemias represents a valuable resource in that germline DNA sequencing has allowed the unambiguous determination of somatic mutations in both PT and PDX.

Characterizing the phenotypic and genetic changes of pre-epidemic HIV-2 group F virus following serial passage in humanized mice.

HIV-2 Group F virus with an origin in NHPs was isolated from only two individuals. Two serial passages in hu-mice showed increased viral loads, CD4+ T cell decline and nonsynonymous genetic changes showing its capacity for further evolution, and spread in the human.

Evaluation of Anti-plasmodial Activities of Aqueous Leaf Extracts of Ficus trichocarpa and Lasianthera africana on Chloroquine-sensitive Plasmodium berghei berghei (NK65) Infected Male Albino Mice

This study was designed to evaluate the antiplasmodial activities of aqueous leaf extracts of Ficus trichocarpa and Lasianthera africana on chloroquine-sensitive Plasmodium berghei berghei (NK65) infected male albino mice. P. berghei was obtained from National Institute for Medical Research, Lagos, Nigeria, and maintained in the laboratory by serial passage in mice. Chemo-suppressive, prophylactic, and curative procedures were used to appraise the antiplasmodial potentials of the extracts. A total of 305 albino mice were used for this study. Forty-five albino mice which were divided into 9 groups were used for the acute toxicity study of F. trichocarpa and L. africana. For chemo-suppressive and prophylactic assessment, 180 albino mice were divided into 2 sets of 90 rats grouped into 6 of five albino mice each respectively, while 70 mice were used for the curative assessment. The remaining 10 infected mice served as donor mice. The infected mice were orally treated daily with 5 mg kg‾1 chloroquine 500, 1000, 1500, and 2000 mg kg‾1 bodyweight of F. trichocarpa and L. africana respectively, and a combined extract of F. trichocarpa and L. africana in a 1:1 ratio. The In vivo antiplasmodial effect of L. africana extract against P. berghei infection showed a significant (p˂0.05) dose-dependent schizonticidal activity for the prophylactic test. The percentage suppression of the malarial parasite by F. trichocarpa and L. africana after a 4-day treatment followed a dose-dependent manner and were significantly (p˂0.05) higher than the percentage suppression of groups treated with the combined extracts. Results of the prophylactic study showed that the percentage suppression of the parasite by the aqueous leaf extract of L. africana at each administered dosage was above 30%. It was, therefore, concluded that the leaf extracts of F. trichocarpa and L. africana possess antimalarial potencies when administered individually than as a combined regime and this property could be exploited for antimalarial therapy.

Short-term culture adaptation of Toxoplasma gondii archetypal II and III field isolates affects cystogenic capabilities and modifies virulence in mice

Most Toxoplasma gondii research has been carried out using strains maintained in the laboratory for long periods of time. Long-term passage in mice or cell culture influences T. gondii phenotypic traits such as the capability to produce oocysts in cats and virulence in mice. In this work, we investigated the effect of cell culture adaptation in the short term for recently obtained type II (TgShSp1 (Genotype ToxoDB#3), TgShSp2 (#1), TgShSp3 (#3) and TgShSp16 (#3)) and type III (#2) isolates (TgShSp24 and TgPigSp1). With this purpose, spontaneous and alkaline stress-induced cyst formation in Vero cells during 40 passages, from passage 10 (p10) to 50 (p50), and isolate virulence at p10 versus p50 were studied using a harmonized bioassay method in Swiss/CD1 mice. T. gondii cell culture maintenance showed a drastic loss of spontaneous and induced production of mature cysts after ≈25–30 passages. The TgShSp1, TgShSp16 and TgShSp24 isolates failed to generate spontaneously formed mature cysts at p50. Limited cyst formation was associated with an increase in parasite growth and a shorter lytic cycle. In vitro maintenance also modified T. gondii virulence in mice at p50 with events of exacerbation, increasing cumulative morbidity for TgShSp2 and TgShSp3 isolates and mortality for TgShSp24 and TgPigSp1 isolates, or attenuation, with absence of mortality and severe clinical signs for TgShSp16, and better control of the infection with the lowest parasite and cyst burdens in lungs and brain for the TgShSp1 isolate. The present findings show deep changes in relevant phenotypic traits in laboratory-adapted T. gondii isolates and open new discussion about their use for inferring keys to parasite biology and virulence.

Abstract 4676: Deconvolution of extrinsic and intrinsic factors influencing tumor growth in solid cancer PDX models to support design, analysis and application of PDX-based pharmacology studies

Abstract PDX models are one of the gold standard models in preclinical drug development. Their intra- and intertumoral heterogeneity is accepted as a model immanent feature. However, the model performance directly impacts the study design as well as the interpretation of experimental data using those models. To discriminate between intrinsic and extrinsic factors driving heterogeneous tumor growth of PDX models we investigated the model performance of >500 subcutaneously growing PDX models of solid cancer by examining growth curves of control arms run in the framework of > 5000 independent experiments. The relative tumor volume (RTV) over time was correlated with the number of passages in mice, the respective mouse strain, and the applied control vehicle per model as well as across tumor types. The analysis of ten different colon cancer PDX models revealed statistically significant differences between RTV on a specific day across different passages in a specific model as well as across all ten models (Kruskall-Wallis test). However, neither the variance nor the doubling time of the tumor correlated with the number of passages in mice. Based on these results we extended the analysis across six different tumor types with 33 distinct models in six different mouse strains. Again, an influence of the passage number on the tumor volume on a specific experimental day was depicted. The range of mean RTV related to a specific passage were least pronounced on day 7 (71% - 1626%), most notable on day 14 (65% - 4543%) and decreasing again on day 21 (34% - 3633%). In this larger cohort a subset of data points was more similar in performance to each other than any other set. The clustering was based on performance on experimental day 7. This subset received either saline or was untreated. This demonstrates that non-saline ingredients have an evident effect on tumor progression. Due to limited size of the dataset the influence of the mouse strain was evident only on a descriptive level. Whereas the use of different nude mouse strains, such as CD1-, athymic, or NMRI nude, did not affect tumor volume over time, the use of more immunodeficient mice such as SCID, NSG or NOG led to a faster tumor growth in some of the models across different passages. In the next iteration we will analyze the full dataset overcoming current limitations due to small subsets in a specific condition. The actual aim of this project is to better understand and measure tumor heterogeneity in PDX models. Based on these analyses the output of PDX-based studies will be optimized, as we get a better understanding how extrinsic factors (mouse strain and vehicle) and intrinsic factors (passage number) influence tumor growth. In a longer perspective the ability to model tumor volume over time in a specific model will reduce the need of control animals in future experiments, thereby actively supporting the concept of 3R. Citation Format: Mathew Clark, Kanstantsin Lashuk, Edward McGowan, Julia Schueler. Deconvolution of extrinsic and intrinsic factors influencing tumor growth in solid cancer PDX models to support design, analysis and application of PDX-based pharmacology studies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4676.

Abstract 3998: Metronomic gemcitabine plus neratinib effectively inhibits the growth of human Her-2 positive breast cancer cells intracranially implanted into immunodeficient mice

Abstract Metastatic breast cancer is a major cause of death for women in the US, and breast cancers that metastasize to the brain have a particularly poor prognosis. To treat brain metastases of Her-2 positive breast cancers a number of Her-2 targeting drugs are now clinically available, including lapatinib, neratinib and tucatinib, among others. However, in part as a consequence of poor penetration of many drugs into the brain, combinations of standard chemotherapy plus targeted agent have only modest impact on progression free survival for such patients. We have developed preclinical models of human Her-2 positive breast cancer brain metastases to help address these issues. We selected human MDA-MD-361 and BT474 breast cancer cells lines by in vivo passaging in SCID mice, followed by molecular tagging of the derived variants (MDA361R and BT474R respectively), and then the stereotactic implantation of such cells into the brain of female SCID mice. Using luciferase-tagged BT474R we implanted 75,000 cells into the brains of the mice, and thereafter confirmed by luminescence the growth of the cells, which allowed for randomization of the mice to receive control saline (n=7), metronomic gemcitabine (100mg/kg/3days; i.p.; n=8), neratinib (40mg/kg/3 days; p.o.; n=7), or the combination of gemcitabine plus neratinib (n=8), which continued for 5 months largely in the absence of overt toxicity. The gemcitabine plus neratinib combination prolonged survival over the controls (150 days vs 65 days, p<0.5), although eventually all mice succumbed to disease. Tumors cells have been isolated from these therapy-administered mice for further characterization. Neratinib alone did not result in an increase in survival compared to controls, whereas metronomic gemcitabine improved survival but was inferior to the combination. We confirmed these findings using MDA361R tagged with human chorionic gonadotropin (hCG), which allows for the growth of cells to be monitored by assessment of hCG in the mouse urine, and these cells were implanted intracranially into female SCID mice. After urine hCG levels confirmed the presence of growing cells in the brain, the mice were randomized to the above 4 therapies (n=4/group), which showed an increase in survival of the combination compared to controls. These results point to intriguing effects of chemotherapy when used at continuous low (metronomic) doses, that in these studies involved gemcitabine administration every 3 days, and which in these models can be effectively combined with neratinib to suppress the growth of Her-2 positive breast cancer cells implanted into the mouse brain. These results highlight promising combination that could be used to suppress Her-2 positive breast cancer brain metastatic growth, and the developed models should facilitate the study of this subset of breast cancers. Citation Format: Serina Batson, Alejandro Sanchez, Hector M. Padilla, Diana L. Prospero, Brenda Lugo, Valeria V. Lopez, Daniella E. Estrada, Nydia De Avila, Saeedeh Darvishi, Arlene Levario, Shan Man, Ping Xu, Robert S. Kerbel, Guido Bocci, Giulio Francia. Metronomic gemcitabine plus neratinib effectively inhibits the growth of human Her-2 positive breast cancer cells intracranially implanted into immunodeficient mice. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3998.

SARS-CoV-2-related bat virus behavior in human-relevant models sheds light on the origin of COVID-19.

Bat sarbecovirus BANAL-236 is highly related to SARS-CoV-2 and infects human cells, albeit lacking the furin cleavage site in its spike protein. BANAL-236 replicates efficiently and pauci-symptomatically in humanized mice and in macaques, where its tropism is enteric, strongly differing from that of SARS-CoV-2. BANAL-236 infection leads to protection against superinfection by a virulent strain. We find no evidence of antibodies recognizing bat sarbecoviruses in populations in close contact with bats in which the virus was identified, indicating that such spillover infections, if they occur, are rare. Six passages in humanized mice or in human intestinal cells, mimicking putative early spillover events, select adaptive mutations without appearance of a furin cleavage site and no change in virulence. Therefore, acquisition of a furin site in the spike protein is likely a pre-spillover event that did not occur upon replication of a SARS-CoV-2-like bat virus in humans or other animals. Other hypotheses regarding the origin of the SARS-CoV-2 should therefore be evaluated, including the presence of sarbecoviruses carrying a spike with a furin cleavage site in bats.

Streptolysin O Deficiency in Streptococcus pyogenes M1T1 covR/S Mutant Strain Attenuates Virulence in In Vitro and In Vivo Infection Models.

Mutation within the Streptococcus pyogenes (Streptococcus group A; Strep A) covR/S regulatory system has been associated with a hypervirulent phenotype resulting from the upregulation of several virulence factors, including the pore-forming toxin, streptolysin O (SLO). In this study, we utilized a range of covR/S mutants, including M1T1 clonal strains (5448 and a covS mutant generated through mouse passage designated 5448AP), to investigate the contribution of SLO to the pathogenesis of covR/S mutant Strep A disease. Up-regulation of slo in 5448AP resulted in increased SLO-mediated hemolysis, decreased dendritic cell (DC) viability post coculture with Strep A, and increased production of tumor necrosis factor (TNF) and monocyte chemoattractant protein 1 (MCP-1) by DCs. Mouse passage of an isogenic 5448 slo-deletion mutant resulted in recovery of several covR/S mutants within the 5448Δslo background. Passage also introduced mutations in non-covR/S genes, but these were considered to have no impact on virulence. Although slo-deficient mutants exhibited the characteristic covR/S-controlled virulence factor upregulation, these mutants caused increased DC viability with reduced inflammatory cytokine production by infected DCs. In vivo, slo expression correlated with decreased DC numbers in infected murine skin and significant bacteremia by 3 days postinfection, with severe pathology at the infection site. Conversely, the absence of slo in the infecting strain (covR/S mutant or wild-type) resulted in detection of DCs in the skin and attenuated virulence in a murine model of pyoderma. slo-sufficient and -deficient covR/S mutants were susceptible to immune clearance mediated by a combination vaccine consisting of a conserved M protein peptide and a peptide from the CXC chemokine protease SpyCEP. IMPORTANCE Streptococcus pyogenes is responsible for significant numbers of invasive and noninvasive infections which cause significant morbidity and mortality globally. Strep A isolates with mutations in the covR/S system display greater propensity to cause severe invasive diseases, which are responsible for more than 163,000 deaths each year. This is due to the upregulation of virulence factors, including the pore-forming toxin streptolysin O. Utilizing covR/S and slo-knockout mutants, we investigated the role of SLO in virulence. We found that SLO alters interactions with host cell populations and increases Strep A viability at sterile sites of the host, such as the blood, and that its absence results in significantly less virulence. This work underscores the importance of SLO in Strep A virulence while highlighting the complex nature of Strep A pathogenesis. This improved insight into host-pathogen interactions will enable a better understanding of host immune evasion mechanisms and inform streptococcal vaccine development programs.

Analysis of Serial Neuroblastoma PDX Passages in Mice Allows the Identification of New Mediators of Neuroblastoma Aggressiveness.

Neuroblastoma is a neural crest cell-derived pediatric tumor characterized by high inter- and intra-tumor heterogeneity, and by a poor outcome in advanced stages. Patient-derived xenografts (PDXs) have been shown to be useful models for preserving and expanding original patient biopsies in vivo, and for studying neuroblastoma biology in a more physiological setting. The maintenance of genetic, histologic, and phenotypic characteristics of the original biopsy along serial PDX passages in mice is a major concern regarding this model. Here we analyze consecutive PDX passages in mice, at both transcriptomic and histological levels, in order to identify potential changes or highlight similarities to the primary sample. We studied temporal changes using mRNA and miRNA expression and correlate those with neuroblastoma aggressiveness using patient-derived databases. We observed a shortening of tumor onset and an increase in proliferative potential in the PDXs along serial passages. This behavior correlates with changes in the expression of genes related to cell proliferation and neuronal differentiation, including signaling pathways described as relevant for neuroblastoma malignancy. We also identified new genes and miRNAs that can be used to stratify patients according to survival, and which could be potential new players in neuroblastoma aggressiveness. Our results highlight the usefulness of the PDX neuroblastoma model and reflect phenotypic changes that might be occurring in the mouse environment. These findings could be useful for understanding the progression of tumor aggressiveness in this pathology.

Integrative Multi-OMICs Identifies Therapeutic Response Biomarkers and Confirms Fidelity of Clinically Annotated, Serially Passaged Patient-Derived Xenografts Established from Primary and Metastatic Pediatric and AYA Solid Tumors.

Establishment of clinically annotated, molecularly characterized, patient-derived xenografts (PDXs) from treatment-naïve and pretreated patients provides a platform to test precision genomics-guided therapies. An integrated multi-OMICS pipeline was developed to identify cancer-associated pathways and evaluate stability of molecular signatures in a panel of pediatric and AYA PDXs following serial passaging in mice. Original solid tumor samples and their corresponding PDXs were evaluated by whole-genome sequencing, RNA-seq, immunoblotting, pathway enrichment analyses, and the drug−gene interaction database to identify as well as cross-validate actionable targets in patients with sarcomas or Wilms tumors. While some divergence between original tumor and the respective PDX was evident, majority of alterations were not functionally impactful, and oncogenic pathway activation was maintained following serial passaging. CDK4/6 and BETs were prioritized as biomarkers of therapeutic response in osteosarcoma PDXs with pertinent molecular signatures. Inhibition of CDK4/6 or BETs decreased osteosarcoma PDX growth (two-way ANOVA, p < 0.05) confirming mechanistic involvement in growth. Linking patient treatment history with molecular and efficacy data in PDX will provide a strong rationale for targeted therapy and improve our understanding of which therapy is most beneficial in patients at diagnosis and in those already exposed to therapy.