Host Mice Research Articles

Physiological Modeling of the Vascularized Human Lung Organoid.

Studies using human lung organoids (hLO) have focused on differentiation of lung epithelial subtypes into distal alveolar unit. A major question has been whether introducing endothelial cells (EC) and resultant vascularization alter development of hLO. We describe herein a method for vessel infiltration of hLO in which we determined differences of these hLOs with standard avascular hLOs. hLO are generated by combining hiPSC-derived lung progenitor cells (LP) with EC at different LP:EC ratios. This results in vascularization of hLO and enables comparisons with hLO generated without EC. We observe red blood-filled vessels in hLOs generated post-implantation into the kidney capsule of NOD/SCID mice. Both human and mouse EC conjoin in the capsule to form chimeric vessels in hLOs. Vessel-infiltrating hLOs show robust generation of alveolar type II epithelial cells (ATII) and alveolar type I cells (ATI), although there was no difference in the observed 1:1 ATII/ATI cell ratio. Electron microscopy revealed better-developed surfactant production apparatus in ATII of vascularized hLOs compared to avascular hLOs. We observed prominent primitive airway sacs with alveolar epithelial cells lining lumen in vascularized vs. avascular hLOs. The vessel-infiltrating hLOs also mounted a robust inflammatory response characterized by mouse PMN influx after challenging host mice with lipopolysaccharide. Thus, interaction of EC with LP generated vascularized hLOs and drive ATII and ATI differentiation and hLOs also mount a robust inflammatory response upon LPS challenge of hLO-transplanted recipient mice. Our results show usefulness of generating hLOs in studying human lung development and mechanisms underlying inflammatory lung injury.

A hybrid method for discovering interferon-gamma inducing peptides in human and mouse

Interferon-gamma (IFN-γ) is a versatile pleiotropic cytokine essential for both innate and adaptive immune responses. It exhibits both pro-inflammatory and anti-inflammatory properties, making it a promising therapeutic candidate for treating various infectious diseases and cancers. We present IFNepitope2, a host-specific technique to annotate IFN-γ inducing peptides, it is an updated version of IFNepitope introduced by Dhanda et al. In this study, dataset used for developing prediction method contain experimentally validated 25,492 and 7983 IFN-γ inducing peptides in human and mouse host, respectively. In initial phase, machine learning techniques have been exploited to develop classification model using wide range of peptide features. Further, to improve machine learning based models or alignment free models, we explore potential of similarity-based technique BLAST. Finally, a hybrid model has been developed that combine best machine learning based model with BLAST. In most of the case, models based on extra tree perform better than other machine learning techniques. In case of peptide features, compositional feature particularly dipeptide composition performs better than one-hot encoding or binary profile. Our best machine learning based models achieved AUROC 0.89 and 0.83 for human and mouse host, respectively. The hybrid model achieved the AUROC 0.90 and 0.85 for human and mouse host, respectively. All models have been evaluated on an independent/validation dataset not used for training or testing these models. Newly developed method performs better than existing method on independent dataset. The major objective of this study is to predict, design and scan IFN-γ inducing peptides, thus server/software have been developed (https://webs.iiitd.edu.in/raghava/ifnepitope2/). This method is also available as standalone at https://github.com/raghavagps/ifnepitope2 and python package index at https://pypi.org/project/ifnepitope2/.

A Retrospective Report on the Infestation and Distribution of Chiggers on an Endemic Rodent Species (Apodemus latronum) in Southwest China

Chiggers are the exclusive vector of Orientia tsutsugamushi, which is the causative agent of scrub typhus. Based on the field surveys in southwest China between 2001 and 2022, this paper retrospectively reported the chigger infestation and distribution on the large-eared field mouse (Apodemus latronum), an endemic rodent species in the region, for the first time. A total of 933 chiggers were collected from 501 mice, and these chiggers were identified as 2 families, 8 genera, and 61 species. The overall infestation prevalence (PM) and mean abundance (MA) of A. latronum with chiggers reached 19.76% and 1.86 mites/per mouse, respectively. The chigger infestation indices on adult A. latronum (PM = 38.28%, MA = 5.11) were higher than those on juvenile mice (PM = 12.63%, MA = 0.97) with p < 0.01, showing an age bias of infestation. The relative fatness (K) was used to reflect the nutrition status of the mouse host. The mouse hosts with good nutrition (K = 3.4 ± 0.89 g/cm3) harbored fewer chiggers than the hosts with poor nutrition (K = 2.2 ± 0.90 g/cm3) (p < 0.01). The infestation indices of chiggers on A. latronum obviously fluctuated along different altitude gradients (p < 0.01). With the increase in altitudes, the β diversity of the chigger community showed a gradually increasing tendency. The spillover chord diagram, which was based on indices of PAC (potential for apparent competition), revealed high spillover potentials of dominant chigger species dispersing from high altitude gradients to the lowest one. The chigger abundance was positively correlated with the mean monthly temperature (tmp), mean monthly humidity (hum), the mean monthly precipitation (pre), and the human footprint (hfp), and it was negatively correlated with the altitude (ele) (p < 0.05). The temperature and humidity are the most important factors which influence the chigger infestation.

In Vitro Vascularization Improves In Vivo Functionality of Human Engineered Cardiac Tissues

Engineered human cardiac tissues hold great promise for disease modeling, drug development, and regenerative therapy. For regenerative applications, successful engineered tissue engraftment in vivo requires rapid vascularization and blood perfusion post-implantation. In the present study, we engineered highly functional, vascularized cardiac tissues (“cardiopatches”) by co-culturing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and endothelial cells (hiPSC-ECs) in optimized serum-free media. The vascularized cardiopatches displayed stable capillary networks over 4 weeks of culture, the longest reported in the field, while maintaining high contractile stress (>15 mN/mm2) and fast conduction velocity (>20 cm/s). Robustness of the method was confirmed using two distinct hiPSC-EC sources. Upon implantation into dorsal-skinfold chambers in immunocompromised mice, in vitro vascularized cardiopatches exhibited improved angiogenesis compared to avascular implants. Significant lumenization of the engineered human vasculature and anastomosis with host mouse vessels yielded the formation of hybrid human-mouse capillaries and robust cardiopatch perfusion by blood. Moreover, compared to avascular tissues, the implanted vascularized cardiopatches exhibited significantly higher conduction velocity and Ca2+ transient amplitude, longitudinally monitored in live mice for the first time. Overall, we demonstrate successful 4-week vascularization of engineered human cardiac tissues without loss of function in vitro, which promotes tissue functionality upon implantation in vivo. Statement of SignificanceComplex interactions between cardiac muscle fibers and surrounding capillaries are critical for everyday function of the heart. Tissue engineering is a powerful method to recreate functional cardiac muscle and its vascular network, which are both lost during a heart attack. Our study demonstrates in vitro engineering of dense capillary networks within highly functional engineered heart tissues that successfully maintain the structure, electrical, and mechanical function long-term. In mice, human capillaries from these engineered tissues integrate with host mouse capillaries to allow blood perfusion and support improved implant function. In the future, the developed vascularized engineered heart tissues will be used for in vitro studies of cardiac development and disease and as a potential regenerative therapy for heart attack.

Species barrier as molecular basis for adaptation of synthetic prions with N-terminally truncated PrP.

Mammalian prions are neurotropic pathogens formed from PrPSc assemblies, a misfolded variant of the host-encoded prion protein PrPC. Multiple PrPSc conformations or strains self-propagate in host populations or mouse models of prion diseases, exhibiting distinct biological and biochemical phenotypes. Constrained interactions between PrPSc and PrPC conformations confer species specificity and regulate cross-species transmission. The pathogenicity of fibrillar assemblies derived from bacterially expressed recombinant PrP (rPrP) has been instrumental in demonstrating the protein-only nature of prions. Yet, their ability to encode different strains and transmit between species remains poorly studied, hampering their use in exploring structure-to-strain relationships. Fibrillar assemblies from rPrP with hamster, mouse, human, and bovine primary structures were generated and tested for transmission and adaptation in tg7 transgenic mice expressing hamster PrPC. All assemblies, except the bovine ones, were fully pathogenic on the primary passage, causing clinical disease, PrPSc brain deposition, and spongiform degeneration. They exhibited divergent adaptation processes and strain properties upon subsequent passage. Assemblies of hamster origin propagated without apparent need for adaptation, those of mouse origin adapted abruptly, and those of human origin required serial passages for optimal fitness. Molecular analyses revealed the presence of endogenously truncated PrPSc species in the resulting synthetic strains that lack the 90-140 amino acid region considered crucial for infectivity. In conclusion, rPrP assemblies provide a facile means of generating novel prion strains with adaptative/evolutive properties mimicking genuine prions. The PrP amino acid backbone is sufficient to encode different strains with specific adaptative properties, offering insights into prion transmission and strain diversity.

Loss of endogenous Nox2-NADPH oxidase does not prevent age-induced platelet activation and arterial thrombosis in mice

BackgroundReactive oxygen species are known to contribute to platelet hyperactivation and thrombosis during aging; however, the mechanistic contribution of the specific oxidative pathway remains elusive. ObjectivesWe hypothesized that during aging, endogenous Nox2-NADPH oxidase contributes to platelet reactive oxygen species accumulation and that loss of Nox2 will protect from platelet activation and thrombosis. MethodsWe studied littermates of Nox2 knockout (Nox2-KO) and -wild-type (Nox2-WT) mice at young (3-4 months) and old (18-20 months) age. Within platelets, we examined the expression of subunits of NADPH oxidase and enzyme activity, oxidant levels, activation markers, aggregation, and secretion. We also assessed susceptibility to in vivo thrombosis in 2 experimental models. ResultsWhile aged Nox2-WT mice displayed increased mRNA levels for Nox2, aged Nox2-KO mice showed an increase in Nox4 mRNA. However, neither the protein levels of several subunits nor the activity of NADPH oxidase were found to be altered by age or genotype. Both aged Nox2-WT and aged Nox2-KO mice exhibited similar enhancement in levels of platelet oxidants, granule release, αIIbβ3 activation, annexin V binding, aggregation and secretion, and a greater susceptibility to platelet-induced pulmonary thrombosis compared with young mice. In a photochemical injury model, adoptive transfer of platelets from aged Nox2-WT or Nox2-KO mice to the aged host mice resulted in a similar time to develop occlusive thrombus in the carotid artery. These findings suggest that loss of endogenous Nox2 does not protect against age-related platelet activation and arterial thrombosis in mice. ConclusionWe conclude that Nox2 is not an essential mediator of prothrombotic effects associated with aging.

Dipeptide metabolite, glutamyl-glutamate mediates microbe-host interaction to boost spermatogenesis

The decrease in sperm count and infertility is a global issue that remains unresolved. By screening environmental bacterial isolates, we have found that a novel lactic acid bacterium, Lactiplantibacillus plantarum SNI3, increased testis size, testosterone levels, sperm count, sexual activity and fertility in mice that have consumed the bacteria for four weeks. The abundance of L. plantarum in the colon microbiome was positively associated with sperm count. Fecal microbiota transplantation (FMT) from L. plantarum SNI3-dosed mice improved testicular functions in microbiome-attenuated recipient animals. To identify mediators that confer pro-reproductive effects on the host, untargeted in situ mass spectrometry metabolomics was performed on testis samples of L. plantarum SNI3-treated and control mice. Enrichment pathway analysis revealed several perturbed metabolic pathways in the testis of treated mice. Within the testis, a dipeptide, glutamyl-glutamate (GluGlu) was the most upregulated metabolite following L. plantarum SNI3 administration. To validate the pro-reproductive feature of GluGlu, systemic and local injections of the dipeptide have been performed. γ-GluGlu increased sperm count but had no effect on testosterone. These findings highlight the role of γ-GluGlu in mediating spermatogenetic effects of L. plantarum on the male mouse host and –following relevant human clinical trials- may provide future tools for treating certain forms of male infertility.

The cellular prion protein does not affect tau seeding and spreading of sarkosyl-insoluble fractions from Alzheimer’s disease

The cellular prion protein (PrPC) plays many roles in the developing and adult brain. In addition, PrPC binds to several amyloids in oligomeric and prefibrillar forms and may act as a putative receptor of abnormal misfolded protein species. The role of PrPC in tau seeding and spreading is not known. In the present study, we have inoculated well-characterized sarkosyl-insoluble fractions of sporadic Alzheimer’s disease (sAD) into the brain of adult wild-type mice (Prnp+/+), Prnp0/0 (ZH3 strain) mice, and mice over-expressing the secreted form of PrPC lacking their GPI anchor (Tg44 strain). Phospho-tau (ptau) seeding and spreading involving neurons and oligodendrocytes were observed three and six months after inoculation. 3Rtau and 4Rtau deposits from the host tau, as revealed by inoculating Mapt0/0 mice and by using specific anti-mouse and anti-human tau antibodies suggest modulation of exon 10 splicing of the host mouse Mapt gene elicited by exogenous sAD-tau. However, no tau seeding and spreading differences were observed among Prnp genotypes. Our results show that PrPC does not affect tau seeding and spreading in vivo.

Reverse vaccinology approaches to design a potent multiepitope vaccine against the HIV whole genome: immunoinformatic, bioinformatics, and molecular dynamics approaches

Substantial advances have been made in the development of promising HIV vaccines to eliminate HIV-1 infection. For the first time, one hundred of the most submitted HIV subtypes and CRFs were retrieved from the LANL database, and the consensus sequences of the eleven HIV proteins were obtained to design vaccines for human and mouse hosts. By using various servers and filters, highly qualified B-cell epitopes, as well as HTL and CD8 + epitopes that were common between mouse and human alleles and were also located in the conserved domains of HIV proteins, were considered in the vaccine constructs. With 90% coverage worldwide, the human vaccine model covers a diverse allelic population, making it widely available. Codon optimization and in silico cloning in prokaryotic and eukaryotic vectors guarantee high expression of the vaccine models in human and E. coli hosts. Molecular dynamics confirmed the stable interaction of the vaccine constructs with TLR3, TLR4, and TLR9, leading to a substantial immunogenic response to the designed vaccine. Vaccine models effectively target the humoral and cellular immune systems in humans and mice; however, experimental validation is needed to confirm these findings in silico.

Determinants of pegivirus persistence, cross-species infection, and adaptation in the laboratory mouse.

Viruses capable of causing persistent infection have developed sophisticated mechanisms for evading host immunity, and understanding these processes can reveal novel features of the host immune system. One such virus, human pegivirus (HPgV), infects ~15% of the global human population, but little is known about its biology beyond the fact that it does not cause overt disease. We passaged a pegivirus isolate of feral brown rats (RPgV) in immunodeficient laboratory mice to develop a mouse-adapted virus (maPgV) that established persistent high-titer infection in a majority of wild-type laboratory mice. maRPgV viremia was detected in the blood of mice for >300 days without apparent disease, closely recapitulating the hallmarks of HPgV infection in humans. We found a pro-viral role for type-I interferon in chronic infection; a lack of PD-1-mediated tolerance to PgV infection; and multiple mechanisms by which PgV immunity can be achieved by an immunocompetent host. These data indicate that the PgV immune evasion strategy has aspects that are both common and unique among persistent viral infections. The creation of maPgV represents the first PgV infection model in wild-type mice, thus opening the entire toolkit of the mouse host to enable further investigation of this persistent RNA virus infections.

Dietary sodium modulates mTORC1-dependent trained immunity in macrophages to accelerate CKD development

Chronic Kidney Disease (CKD) is a significant global health issue linked to dietary habits, especially high salt intake. However, the precise mechanisms driving this progression remain incompletely understood. This study reveals that a high-salt diet intensifies macrophage trained immunity, leading to a marked pro-inflammatory response upon repeated pathogenic exposures, as evidenced by increased renal damage and fibrosis. Under high-salt conditions, there was an induction of CD45+F4/80+ macrophage infiltration into the renal tissue, accompanied by heightened production of inflammatory cytokines. Distinct responses were observed between circulating and resident renal macrophages to a high-salt diet, with a notable upsurge in the migration of pro-inflammatory macrophages, driven by CCL2-CCR2 signaling and aberrant mTORC1 pathway activation. Treatment with rapamycin-liposome effectively reduced this inflammatory cascade by mitigating mTORC1 signaling. Transplantation of monocytes from CKD mice with a high-salt diet significantly exacerbates renal inflammatory damage in the host mice, showing increased migratory tendency and inflammatory activity. The cell co-culture experiment further confirmed that macrophages derived from CKD mice, particularly those under conditions of high salt exposure, significantly induced apoptosis and inflammatory responses in renal tubular cells. Taken together, recurrent exposure to LPS elicits the activation of trained immunity, consequently augmenting inflammatory response of monocytes/macrophages in the involved kidneys. The high-salt diet exacerbates this phenomenon, attributable at least in part to the overactivation of the mTORC1 pathway. This research emphasizes the importance of dietary modulation and targeted immunological interventions in slowing CKD progression, providing new insights into mTORC1-mediated pathophysiological mechanisms and potential management strategies for CKD.

Enteric tuft cells coordinate timely expulsion of the tapeworm Hymenolepis diminuta from the murine host by coordinating local but not systemic immunity.

Recognizing that enteric tuft cells can signal the presence of nematode parasites, we investigated whether tuft cells are required for the expulsion of the cestode, Hymenolepis diminuta, from the non-permissive mouse host, and in concomitant anti-helminthic responses. BALB/c and C57BL/6 mice infected with H. diminuta expelled the worms by 11 days post-infection (dpi) and displayed DCLK1+ (doublecortin-like kinase 1) tuft cell hyperplasia in the small intestine (not the colon) at 11 dpi. This tuft cell hyperplasia was dependent on IL-4Rα signalling and adaptive immunity, but not the microbiota. Expulsion of H. diminuta was slowed until at least 14 dpi, but not negated, in tuft cell-deficient Pou2f3-/- mice and was accompanied by delayed goblet cell hyperplasia and slowed small bowel transit. Worm antigen and mitogen evoked production of IL-4 and IL-10 by splenocytes from wild-type and Pou2f3-/- mice was not appreciably different, suggesting similar systemic immune reactivity to infection with H. diminuta. Wild-type and Pou2f3-/- mice infected with H. diminuta displayed partial protection against subsequent infection with the nematode Heligmosomoides bakeri. We speculate that, with respect to H. diminuta, enteric tuft cells are important for local immune events driving the rapidity of H. diminuta expulsion but are not critical in initiating or sustaining systemic Th2 responses that provide concomitant immunity against secondary infection with H. bakeri.

N-ethyl-N-nitrosourea (ENU)-induced C-terminal truncation of Runx3 results in autoimmune colitis associated with Th17/Treg imbalance

Inflammatory bowel disease (IBD) is a chronic and progressive inflammatory intestinal disease that affects people around the world. The primary cause of IBD is an imbalance in the host immune response to intestinal flora. Several human genes, including IL10, STAT3, IRGM, ATG16L1, NOD2 and RUNX3, are associated with inappropriate immune responses in IBD. It has been reported that homozygous Runx3-knockout (ko) mice spontaneously develop colitis. However, the high mortality rate in these mice within the first two weeks makes it challenging to study the role of Runx3 in colitis. To address this issue, a spontaneous colitis (SC) mouse model carrying a C-terminal truncated form of Runx3 with Tyr319stop point mutation has been generated. After weaning, SC mice developed spontaneous diarrhea and exhibited prominent enlargement of the colon, accompanied by severe inflammatory cell infiltration. Results of immunofluorescence staining showed massive CD4+ T cell infiltration in the inflammatory colon of SC mice. Colonic IL-17A mRNA expression and serum IL-17A level were increased in SC mice. CD4+ T cells from SC mice produced stronger IL-17A than those from wildtype mice in Th17-skewing conditions in vitro. In addition, the percentages of Foxp3+ Treg cells as well as the RORγt+Foxp3+ Treg subset, known for its role in suppressing Th17 response in the gut, were notably lower in colon lamina propria of SC mice than those in WT mice. Furthermore, transfer of total CD4+ T cells from SC mice, but not from wildtype mice, into Rag1-ko host mice resulted in severe autoimmune colitis. In conclusion, the C-terminal truncated Runx3 caused autoimmune colitis associated with Th17/Treg imbalance. The SC mouse model is a feasible approach to investigate the effect of immune response on spontaneous colitis.

Contribution of parasite and host genotype to immunopathology of schistosome infections

BackgroundThe role of pathogen genotype in determining disease severity and immunopathology has been studied intensively in microbial pathogens including bacteria, fungi, protozoa and viruses but is poorly understood in parasitic helminths. The medically important blood fluke Schistosoma mansoni is an excellent model system to study the impact of helminth genetic variation on immunopathology. Our laboratory has demonstrated that laboratory schistosome populations differ in sporocyst growth and cercarial production in the intermediate snail host and worm establishment and fecundity in the vertebrate host. Here, we (i) investigate the hypothesis that schistosome genotype plays a significant role in immunopathology and related parasite life history traits in the vertebrate mouse host and (ii) quantify the relative impact of parasite and host genetics on infection outcomes.MethodsWe infected BALB/c and C57BL/6 mice with four different laboratory schistosome populations from Africa and the Americas. We quantified disease progression in the vertebrate host by measuring body weight and complete blood count (CBC) with differential over a 12-week infection period. On sacrifice, we assessed parasitological (egg and worm counts, fecundity), immunopathological (organ measurements and histopathology) and immunological (CBC with differential and cytokine profiles) characteristics to determine the impact of parasite and host genetics.ResultsWe found significant variation between parasite populations in worm numbers, fecundity, liver and intestine egg counts, liver and spleen weight, and fibrotic area but not in granuloma size. Variation in organ weight was explained by egg burden and intrinsic parasite factors independent of egg burden. We found significant variation between infected mouse lines in cytokine levels (IFN-γ, TNF-α), eosinophils, lymphocytes and monocyte counts.ConclusionsThis study showed that both parasite and host genotype impact the outcome of infection. While host genotype explains most of the variation in immunological traits, parasite genotype explains most of the variation in parasitological traits, and both host and parasite genotypes impact immunopathology outcomes.Graphical

Maintenance of muscle fiber-type memory in a human-to-mouse muscle xenograft model

INTRODUCTION. Muscle tissue can regenerate through muscle stem cells known as muscle satellite cells (MuSCs). MIME is a technique developed in our laboratory, through which, donor muscle tissue with MuSCs can be implanted into a host muscle to promote donor-cell-derived myogenesis. OBJECTIVE. We studied whether or not MuSCs maintain memory of the type of muscle fibers (fast or slow) with which they were associated. We hypothesized that MuSCs do not maintain memory of the fibers with which they are associated and form slow or fast muscle fibers based on the host environment. METHODS. We tested our hypothesis by implanting donor muscle, which contained mostly slow fibers, into host muscle, which contained mostly fast fibers. Implantation of donor tissue was performed by MIME. Following MIME, we injected a myotoxin (1.2% barium chloride, BaCl2) into the host muscle, which induced concerted degeneration followed by MuSC-dependent regeneration. Specifically, we studied mouse tibialis anterior muscles, which were implanted with segments of human donor tibialis anterior muscle. The human TA is comprised mostly of slow fibers (~60%; i.e., slow myosin heavy chain positive [sMyHC+]). The mouse TA is comprised 100% of fast fibers (i.e., fast myosin heavy chain positive [fMyHC+] fibers). This experimental paradigm is appropriate to ascertain whether or not satellite cells retain memory of the muscle fibers with which they were originally associated. We collected host muscles after 12 weeks of regeneration to ascertain if there was an increase in the percentage of slow muscle fibers. We used antibodies specific to sMyHC and fMyHC to stain frozen TA muscle sections. Host mice were immunodeficient and ubiquitously expressed enhanced green fluorescent protein (NSG-EGFP, The Jackson Laboratory Strain #021937). Since host mouse muscle fibers exhibited green fluorescence, but donor human muscle fibers did not, host and donor muscle fibers could be easily differentiated under fluorescence optics. RESULTS. Quantitative data confirmed that the cluster of human muscle fibers, which regenerated in the host mouse TA muscle had more slow (~50%) than fast (~30%) fibers (T-test, p<0.01, N = 4 TA muscles from 4 different host mice). CONCLUSION. Contrary to our hypothesis, our data suggest that MuSCs do maintain memory of the type of muscle fibers with which they were originally associated. MuSCs give rise to fast or slow muscle fibers based on their donor origin when they are implanted along with their original donor muscle tissue. This work was supported by NIH R03HD091648, Pitch Award from the Alliance for Regenerative Rehabilitation Research and Training (AR3T) from NIH P2CHD086843, and a subcontract from NIH R01AR079884-01 (Peter L. Jones PI) to JAR. We thank the Wayne State University DPT Program for supporting student research and providing research training through coursework and research experiences. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract LB355: Cryo-immune vaccination (CIV) by SYNC-T therapy: Preclinical modeling of a novel device-multidrug immunotherapeutic approach to eradicate advanced metastatic cancers

Abstract A major challenge to further development of immune checkpoint therapies (ICT) for advanced cancer is the inherent toxicity of higher-order multidrug combinations likely needed to eradicate metastatic disease. Recently, early clinical studies have investigated a new general approach to address this challenge, termed cryo-immune vaccination (CIV). The CIV approach by SYNC-T therapy involves cycles of local cryolysis of a solid tumor followed by infusion of the same lesion with a low-dose multi-API formulation. CIV has never been explored methodically in preclinical or clinical settings, so the basis for systemic responses that have been observed in early clinical testing are not fully understood. Accordingly, as trials of the safety and efficacy of CIV proceed, there is now a need of preclinical model systems to help illuminate the mechanistic foundations of the approach, investigate safety and efficacy, and learn where and how it might be best leveraged. To develop the conceptual framework of CIV, we adapted the orthotopic metastatic 4T1 breast cancer model used extensively in our laboratory as a foundation to study CIV antitumor responses and mechanisms of action. Briefly, breast tumors were initiated in naïve murine subjects by orthotopic injection of 1x104 cells into the mammary fad pads of immunocompetent BALB/c hosts. When tumors reached ~5-7 mm, a time when occult pulmonary metastases have seeded, primary tumors were exposed surgically on skin flaps and contacted directly with a liquid nitrogen-cooled pointed steel rod for 15-30 sec. The rod was then withdrawn and tumor tissue allowed to thaw fully before injection into the cryolysed tumor of a selected ICT multidrug cocktail (or negative control murine IgG) in 25-50 µl total volume. The selected multidrug cocktail was modeled on SYNC-T combination therapy currently under study in a Phase 1 clinical trial (Syncromune, Inc.) Briefly, the ICT drug cocktails were variably composed of murine-reactive antibodies against CTLA4, PD-1 or OX40 (agonist), CD40L (agonist) and CpG oligonucleotide. As a starting point, allometric scaling was used to model dosing analogous to the proprietary ICT drug formulations SV-101 and SV-102 in clinical testing (Syncromune, Inc.). For exploratory toxicology, a standard panel of blood-borne liver and kidney toxicity biomarkers was assessed. For exploratory pharmacology, a ‘super mouse’ design was employed with ELISA-based determinations to measure antibody distribution in blood serum. To gain initial mechanistic insights, responses will be compared in host mice that are genetically deficient in selected components of the adaptive or innate immune systems. Progress in methodological and mechanistic studies will be presented. Citation Format: James B. DuHadaway, Alexander J. Muller, Lisa D. Laury-Kleintop, U. Margaretha Wallon, Susan K. Gilmour, Marie Webster, Jason R. Williams, Gabriela R. Rossi, Mario R. Mautino, Jonathan Lewis, Charles J. Link, George C. Prendergast. Cryo-immune vaccination (CIV) by SYNC-T therapy: Preclinical modeling of a novel device-multidrug immunotherapeutic approach to eradicate advanced metastatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB355.

A compendium of multi-omics data illuminating host responses to lethal human virus infections

Human infections caused by viral pathogens trigger a complex gamut of host responses that limit disease, resolve infection, generate immunity, and contribute to severe disease or death. Here, we present experimental methods and multi-omics data capture approaches representing the global host response to infection generated from 45 individual experiments involving human viruses from the Orthomyxoviridae, Filoviridae, Flaviviridae, and Coronaviridae families. Analogous experimental designs were implemented across human or mouse host model systems, longitudinal samples were collected over defined time courses, and global multi-omics data (transcriptomics, proteomics, metabolomics, and lipidomics) were acquired by microarray, RNA sequencing, or mass spectrometry analyses. For comparison, we have included transcriptomics datasets from cells treated with type I and type II human interferon. Raw multi-omics data and metadata were deposited in public repositories, and we provide a central location linking the raw data with experimental metadata and ready-to-use, quality-controlled, statistically processed multi-omics datasets not previously available in any public repository. This compendium of infection-induced host response data for reuse will be useful for those endeavouring to understand viral disease pathophysiology and network biology.

Generation of rat forebrain tissues in mice

Interspecies blastocyst complementation (IBC) provides a unique platform to study development and holds the potential to overcome worldwide organ shortages. Despite recent successes, brain tissue has not been achieved through IBC. Here, we developed an optimized IBC strategy based on C-CRISPR, which facilitated rapid screening of candidate genes and identified that Hesx1 deficiency supported the generation of rat forebrain tissue in mice via IBC. Xenogeneic rat forebrain tissues in adult mice were structurally and functionally intact. Cross-species comparative analyses revealed that rat forebrain tissues developed at the same pace as the mouse host but maintained rat-like transcriptome profiles. The chimeric rate of rat cells gradually decreased as development progressed, suggesting xenogeneic barriers during mid-to-late pre-natal development. Interspecies forebrain complementation opens the door for studying evolutionarily conserved and divergent mechanisms underlying brain development and cognitive function. The C-CRISPR-based IBC strategy holds great potential to broaden the study and application of interspecies organogenesis.

Abstract 2758: Stemness subtype gastric cancer accelerate hematogenous metastasis

Abstract Oncogenic progression and metastasis correlate with the acquisition of stem cell-like characteristics and the loss of differentiated phenotype. Poorly differentiated primary tumors often lead to unfavorable oncologic outcomes due to a heightened likelihood of metastasis to distant organs across various cancers. However, the relationship between stemness traits and metastasis in gastric cancer (GC) remains insufficiently explored. In this investigation, we analyzed bulk RNA-sequencing from microdissected primary tumor specimens and clinical data to pinpoint molecular subtypes linked to distant organ metastasis. Transcriptomic data from patient-derived xenograft (PDX) samples and available single-cell RNA datasets validated our bulk RNAseq findings. Comprehensive analysis highlighted the molecular traits of GC tied to hematogenous metastasis. Microdissection of primary tumor FFPE samples facilitated bulk RNA sequencing, encompassing samples from 33 metastatic and 31 metastasis-free patients, with 8 paired normal samples. Most samples contained less than 10% of tumor microenvironment (TME) components like fibroblasts, endothelial cells, and immune cells. Using non-negative matrix factorization (NMF) clustering, we achieved unsupervised subtyping of the gastric cancer gene expression matrix. Permutation tests based on subsampling indicated consistent high-level cophenetic coefficients at k=2, suggesting robust bifurcation of transcriptomic data into two clusters. One showed augmented stemness traits while the other emphasized differentiated mucosal genes, labeled as stemness and differentiated subtypes, respectively. GSEA highlighted a significant boost in the Hallmark angiogenesis score in the stemness subtype, not attributed to endothelial cell infiltration as verified by CIBERSORTx. This subtype also exhibited a marked decrease in hematogenous metastasis-free survival (HMFS) (p=0.008, log-rank test). Further, PDX samples were scrutinized. Post classification of the PDX graft expression matrix via logistic regression, we examined the endothelial cell fraction, determined by CIBERSORTx. Stemness subtype PDX grafts revealed a significant elevation in the host (mouse) endothelial cell fraction (p<0.05). In addition, stemness PDX patient showed significant decreased HMFS (p = 0.03). We also categorized the pseudobulk expression matrix of malignant cells into stemness or differentiated subtypes using GSE183904 scRNA cohort. scRNA sample analysis consistently presented an elevated endothelial cell fraction in the stemness GC subtype. The outcomes suggest a propensity for angiogenesis in the TME of the stemness subtype of GC. In conclusion, our findings demonstrate that the stemness subtype of GC accelerates hematogenous metastasis, as evidenced through clinical data, bulk RNAseq, PDX, and scRNAseq data. Citation Format: Seungho Lee, Jaeun Yoo, Seungbok Lee, Hyun Myong Kim, Kyoungyun Jeong, Yie-Ri Yoo, Ji-Yeon Shin, Kyoung Un Park, Hye Seung Lee, Seong-Ho Kong, Do Joong Park, Hyuk-Joon Lee, Han-Kwang Yang. Stemness subtype gastric cancer accelerate hematogenous metastasis [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 2758.

Abstract 1540: The dendritic cell receptor DNGR-1 shapes immunity to cancer

Abstract Background: Central to the success of CD8+ T cell-mediated anti-cancer response are type 1 conventional dendritic cells (cDC1s), acting as professional antigen presenting cells. In both mouse and human, DNGR-1 (a.k.a. CLEC9A) is a dedicated cDC1 receptor that, upon binding to its physiological ligand filamentous-actin (F-actin) exposed upon cell death, prime cytotoxic CD8+ T cells specifically against dead cell-associated antigens. Here, we interrogate the role of DNGR-1 in shaping the tumor microenvironment during carcinogenesis. Methods: We utilized two models of chemical carcinogenesis: 3-methylcholanthrene (MCA)-induced sarcoma, and azoxymethane (AOM) and dextran sodium sulfate colitis (DSS)-associated carcinoma. Rag1−/- (no B and T cells), Clec9agfp/gfp, Clec9acre/cre and WT (wildtype) mice on C57BL/6 background were used. Primary MCA-sarcoma cell lines were generated and subsequently tested in a series of transplantation experiments in naïve syngeneic WT mice. Whole exome and RNA sequencing were performed to elucidate the mutational landscape and decipher the immunopeptidome of these primary cell lines. Results: Upon challenge with MCA, there is shorter latency for tumorigenesis in DNGR-1-deficient mice (n=46), like in Rag1−/− mice (n=43), in contrast to immunocompetent WT hosts (n=59) (median: 95 vs 113 days, hazard ratio: 1.72, 95% CI 1.11-2.68, p=0.008). DNGR-1-deficient mice also display increased susceptibility to AOM-DSS carcinogenesis compared to co-housed WT mice. Majority (n=16/24, 68%) of MCA-induced primary sarcoma cell lines derived from DNGR-1-deficient hosts are highly immunogenic and were controlled/rejected at a similar rate, upon challenge in secondary naïve WT recipients, like those derived from Rag1−/− mice (n=13/19, 68%). Whole-exome sequencing of cell lines derived from DNGR-1-deficient hosts revealed that they harbor high mutational burden, including mutations in F-actin-binding proteins that have been previously associated with better survival outcomes in patients with cancer (Giampazolias et al. Cell, 2021). Collectively, these data argue that DNGR-1 protects against carcinogenesis and selects for less immunogenic tumor cells. To evaluate the degree of immunoediting which occurred in the respective primary host mice, we applied the evolutionary metric based on the ratio of nonsynonymous to synonymous mutations (dN/dS), and found that they are less immunoedited compared to cell lines derived from WT hosts. Conclusion: Our findings highlight DNGR-1 as a novel immune checkpoint that couples cell death sensing and (neo-)antigen cross-presentation to functional tumor-specific CD8+ T cell responses during cancer immunoediting. Understanding the mechanism by which DNGR-1 sculpts the mutational landscape of tumors may further inform the dynamics of cancer evolution and may have translational therapeutic implications in directing effective immunotherapy strategies. Citation Format: Kok Haw Lim, Evangelos Giampazolias, Bruno Frederico, Neil C. Rogers, Probir Chakravarty, Gavin Kelly, Ana Cardoso, Emma Nye, Alejandro Suárez-Bonnet, Simon Priestnall, Robert Goldstone, Cell Services Science Technology Platform, Sonia Lee, Oliver Schulz, Jessica Strid, Luis Zapata, Caetano Reis e Sousa. The dendritic cell receptor DNGR-1 shapes immunity to cancer [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 1540.