Discovery of Ketal-Ester Ionizable Lipid Nanoparticle with Reduced Hepatotoxicity, Enhanced Spleen Tropism for mRNA Vaccine Delivery.

The safety and efficacy of the lipid nanoparticle (LNP) delivery system are crucial for the successful development of messenger RNA vaccines. We designed and synthesized a series of ketal ester lipids (KELs), featuring a biodegradable ketal moiety in the linker and ester segments in the tail. Through iterative optimization of the head and tail groups of KELs, we tuned the pKa and molecular shapes, and identified (4S)-KEL12 as a safe and efficient ionizable lipid for mRNA delivery. (4S)-KEL12 LNP showed significantly higher delivery efficacy and lower toxicity than the DLin-MC3-DMA LNP. In comparison to SM-102 LNP, (4S)-KEL12 LNP exhibited better spleen tropism, reduced liver tropism, and hepatotoxicity. Additionally, (4S)-KEL12 demonstrated good biodegradability following intramuscular or intravenous injection. Notably, (4S)-KEL12 LNP encapsulated with a therapeutic mRNA cancer vaccine elicited robust cellular immune responses leading to substantial tumor regression along with prolonged survival in tumor-bearing mice. Our results suggest that (4S)-KEL12 LNP holds great promise for mRNA vaccine delivery. The comprehensive analysis of the structure-activity relationship, toxicity, biodegradability, distribution, expression, efficacy, and stereochemistry of these LNPs will greatly contribute to the rational design and discovery of novel lipid-based delivery systems.

ctDNA whole exome sequencing in pancreatic ductal adenocarcinoma unveils organ-dependent metastatic mechanisms and identifies actionable alterations in fast progressing patients.

Understanding progression mechanisms and developing new targeted therapies is imperative in pancreatic ductal adenocarcinoma (PDAC). In this study, 80 metastatic PDAC patients were prospectively recruited and divided into discovery (n=37) and validation (n=43) cohorts. Tumor and plasma samples taken at diagnosis were pair analyzed using whole exome sequencing (WES) in patients belonging to the discovery cohort alone. The variant allele frequency (VAF) of KRAS mutations was measured by ddPCR in plasma at baseline and response assessment in all patients. Plasma WES identified at least one pathogenic variant across the cohort, uncovering oncogenic mechanisms, DNA repair, microsatellite instability, and alterations in the TGFb pathway. Interestingly, actionable mutations were mostly found in plasma rather than tissue. Patients with shorter survival showed enrichment in cellular organization regulatory pathways. Through WES we could identify a specific molecular profile of patients with liver metastasis, which exhibited exclusive mutations in genes related to the adaptive immune response pathway, highlighting the importance of the immune system in liver metastasis development. Moreover, KRAS mutations in plasma (both at diagnosis and persistent at follow-up) correlated with shorter progression free survival (PFS). Patients presenting a reduction of over 84.75 % in KRAS VAF at response assessment had similar PFS to KRAS-negative patients. Overall, plasma WES reveals molecular profiles indicative of rapid progression, potentially actionable targets, and associations between adaptive immune response pathway alterations and liver tropism.

Glycocalyx-Mimicking Nanoparticles with Differential Organ Selectivity for Drug Delivery and Therapy.

Organ-selective drug delivery is expected to maximize the efficacy of various therapeutic modalities while minimizing their systemic toxicity. Lipid nanoparticles and polymersomes can direct the organ-selective delivery of mRNAs or gene editing machineries, but their delivery is limited to mostly liver, spleen, and lung. A platform that enables delivery to these and other target organs is urgently needed. Here, a library of glycocalyx-mimicking nanoparticles (GlyNPs) comprising five randomly combined sugar moieties is generated, and direct in vivo library screening is used to identify GlyNPs with preferential biodistribution in liver, spleen, lung, kidneys, heart, and brain. Each organ-targeting GlyNP hit show cellular tropism within the organ. Liver, kidney, and spleen-targeting GlyNP hits equipped with therapeutics effectively can alleviate the symptoms of acetaminophen-induced liver injury, cisplatin-induced kidney injury, and immune thrombocytopenia in mice, respectively. Furthermore, the differential organ targeting of GlyNP hits is influenced not by the protein corona but by the sugar moieties displayed on their surface. It is envisioned that the GlyNP-based platform may enable the organ- and cell-targeted delivery of therapeutic cargoes.

Lipid nanovehicles overcome barriers to systemic RNA delivery: Lipid components, fabrication methods, and rational design.

Lipid nanovehicles are currently the most advanced vehicles used for RNA delivery, as demonstrated by the approval of patisiran for amyloidosis therapy in 2018. To illuminate the unique superiority of lipid nanovehicles in RNA delivery, in this review, we first introduce various RNA therapeutics, describe systemic delivery barriers, and explain the lipid components and methods used for lipid nanovehicle preparation. Then, we emphasize crucial advances in lipid nanovehicle design for overcoming barriers to systemic RNA delivery. Finally, the current status and challenges of lipid nanovehicle-based RNA therapeutics in clinical applications are also discussed. Our objective is to provide a comprehensive overview showing how to utilize lipid nanovehicles to overcome multiple barriers to systemic RNA delivery, inspiring the development of more high-performance RNA lipid nanovesicles in the future.

Lymphatic Invasion of Plakoglobin-Dependent Tumor Cell Clusters Drives Formation of Polyclonal Lung Metastases in Colon Cancer.

BACKGROUND & AIMS: Patients with colon cancer with liver metastases may be cured with surgery, but the presence of additional lung metastases often precludes curative treatment. Little is known about the processes driving lung metastasis. This study aimed to elucidate the mechanisms governing lung vs liver metastasis formation. METHODS: Patient-derived organoid (PDO) cultures were established from colon tumors with distinct patterns of metastasis. Mouse models recapitulating metastatic organotropism were created by implanting PDOs into the cecum wall. Optical barcoding was applied to trace the origin and clonal composition of liver and lung metastases. RNA sequencing and immunohistochemistry were used to identify candidate determinants of metastatic organotropism. Genetic, pharmacologic, in vitro, and in vivo modeling strategies identified essential steps in lung metastasis formation. Validation was performed by analyzing patient-derived tissues. RESULTS: Cecum transplantation of 3 distinct PDOs yielded models with distinct metastatic organotropism: liver only, lung only, and liver and lung. Liver metastases were seeded by single cells derived from select clones. Lung metastases were seeded by polyclonal clusters of tumor cells entering the lymphatic vasculature with very limited clonal selection. Lung-specific metastasis was associated with high expression of desmosome markers, including plakoglobin. Plakoglobin deletion abrogated tumor cell cluster formation, lymphatic invasion, and lung metastasis formation. Pharmacologic inhibition of lymphangiogenesis attenuated lung metastasis formation. Primary human colon, rectum, esophagus, and stomach tumors with lung metastases had a higher N-stage and more plakoglobin-expressing intra-lymphatic tumor cell clusters than those without lung metastases. CONCLUSIONS: Lung and liver metastasis formation are fundamentally distinct processes with different evolutionary bottlenecks, seeding entities, and anatomic routing. Polyclonal lung metastases originate from plakoglobin-dependent tumor cell clusters entering the lymphatic vasculature at the primary tumor site.

New Postmortem Perspective on Emerging SARS-CoV-2 Variants of Concern, Germany.

We performed autopsies on persons in Germany who died from COVID-19 and observed higher nasopharyngeal SARS-CoV-2 viral loads for variants of concern (VOC) compared with non-VOC lineages. Pulmonary inflammation and damage appeared higher in non-VOC than VOC lineages until adjusted for vaccination status, suggesting COVID-19 vaccination may mitigate pulmonary damage.

Identification of nine mutant genes and establishment of three prediction models of organ tropism metastases of non-small cell lung cancer.

BACKGROUND: Most Non-small cell lung cancer (NSCLC) patients tend to have metastases at the initial diagnosis. However, limited knowledge has been established regarding which factors, are associated with its metastases. This study aims to identify more biomarkers associated with its organ tropism metastasis and to establish models for prediction of its metastatic organs. METHODS: We performed targeted next-generation sequencing (NGS) to detect genes related to lung cancer in 272 patients with primary advanced NSCLC from Northeast China. We adopted Fisher test, multivariate logistic regression analysis to identify metastasis-related gene mutations and to establish prediction models. RESULTS: Mutations of EGFR (p = 0.0003, OR = 2.554) (especially EGFR L858R [p = 0.02, OR = 2.009]), ATM (p = 0.008, OR = 11.032), and JAK2 (p = 0.009, OR = Inf) were positively and of TP53 exon4mut (p = 0.001, OR = 0.173) was negatively correlated with lung metastasis, and those of CSF1R (p = 0.01, OR = Inf), KIT (p = 0.03, OR = 4.746), MYC (p = 0.05, OR = 7.938), and ERBB2 (p = 0.02, OR = 2.666) were positively correlated with pleural dissemination; those of TP53 (p = 0.01, OR = 0.417) was negatively, while of SMAD4 (p = 0.03, OR = 4.957) was positively correlated with brain metastasis of NSCLC. Additionally, smoking history (p = 0.004, OR = 0.004) was negatively correlated with pleural dissemination of NSCLC. Furthermore, models for prediction of lung metastasis (AUC = 0.706), pleural dissemination (AUC = 0.651), and brane metastasis (AUC = 0.629) were established. CONCLUSION: Taken together, this study revealed nine mutant genes and smoking history associated with organ tropism metastases of NSCLC and provided three models for the prediction of metastatic organs. This study enables us to predict the organs to which non-small cell lung cancer metastasizes before it does develop.

Prevalence and organ tropism of crane-associated adenovirus 1 in cranes overwintering on the Izumi plain, Japan.

Crane-associated adenovirus 1 (CrAdV-1) is a proposed novel virus in the genus Aviadenovirus, first detected in faecal samples from hooded cranes (a vulnerable crane species) on the Izumi plain, a major overwintering site for migratory cranes in Japan. CrAdV-1 was genetically characterized in that study; however, its virological characteristics remain largely unclear. To investigate the prevalence and organ tropism of CrAdV-1, we collected swab and organ samples from dead or debilitated cranes on the Izumi plain. CrAdV-1 gene was detected in 47% (45/95) of tested cranes, comprising mainly hooded cranes but also white-naped and sandhill cranes. These results indicate that CrAdV-1 shedding is widespread among cranes overwintering on the Izumi plain. Phylogenetic analyses revealed that the 68 nucleotide sequences determined from the positive swabs formed a single cluster, suggesting phylogenetic differences between CrAdV-1 and other aviadenoviruses. CrAdV-1 prevalence showed a significant linear increase with time through the overwintering period (November to February), especially among juveniles. These findings indicate that CrAdV-1 spreads mainly by transmission between juveniles progressively through the overwintering period. The CrAdV-1 gene-positive rate was significantly higher in cloacal swabs than conjunctival or tracheal swabs. Copy numbers for the partial CrAdV-1 gene sequence were markedly high in the colon samples from three of the four cranes investigated for organ tropism. We also detected relatively high copy numbers in the cerebrum, trachea, lung and heart, suggesting that CrAdV-1 mainly targets these four organs and transmitted via the faecal-oral route and airborne transmission. These results contribute to further understanding of the virological characteristics of CrAdV-1.

The Road to Dissemination: The Concept of Oligometastases and the Barriers for Widespread Disease.

Over the last years, the oligometastatic disease state has gained more and more interest, and randomized trials are now suggesting an added value of stereotactic radiotherapy on all macroscopic disease in oligometastatic patients; but what barriers could impede widespread disease in some patients? In this review, we first discuss the concept of oligometastatic disease and some examples of clinical evidence. We then explore the route to dissemination: the hurdles a tumoral clone has to overtake before it can produce efficient and widespread dissemination. The spectrum theory argues that the range of metastatic patterns encountered in the clinic is the consequence of gradually obtained metastatic abilities of the tumor cells. Tumor clones can obtain these capabilities by Darwinian evolution, hence early in their genetic progression tumors might produce only a limited number of metastases. We illustrate selective dissemination by discussing organ tropism, the preference of different cancer (sub)types to metastasize to certain organs. Finally we discuss biomarkers that may help to distinguish the oligometastatic state.

Echinococcus granulosus sensu lato in animal intermediate hosts: What is with the organ location?

Organ or tissue tropism/preference in many parasites is essential for their establishment, survival and pathogenesis. In protozoan parasites e.g. Trypanosoma and Plasmodium, tissue tropism is associated with many important aspects such as transmission, treatment, and pathogenesis. In these parasites, tissue tropism is achieved by mechanisms such as sequestration, extravasation, transcellular migration, and vascular permeability. In contrast, little is known about the mechanism behind organ tropism/preference and the potential implication in parasitic metazoans like Echinococcus. Since intraspecies/genetic variation was described for Echinococcus, the organ preference of currently known species and subspecies among animal intermediate hosts remains unclear or perhaps poorly investigated. Here, analysis of 89,359 Echinococcus cysts from different animal intermediate hosts across 47 countries suggests a potential organ distribution pattern in different animal intermediate hosts. For example, E. granulosus s.s. (G1, G3) and E. canadensis (G7) were found more in the liver of sheep and pigs, respectively than in other organs, whereas E. ortleppi and E. canadensis G6 were significantly higher in the lung of cattle and camels, respectively. While the mechanism of organ tropism in Echinococcus and whether it is species/genotype-dependent, host-dependent, or a combination of both is still a subject of future investigations, further and detailed investigation could provide significant information that may be applicable in the diagnosis and treatment of organ-specific cystic echinococcosis or designing host or genotype-specific interventions.

Cancer cell foraging to explain bone-specific metastatic progression.

Metastatic cancer is lethal and patients who suffer bone metastases fare especially poorly. Bone-specific metastatic progression in prostate and breast cancers is a highly observed example of organ-specific metastasis, or organotropism. Though research has delineated the sequential steps of the metastatic cascade, the determinants of bone-specific metastasis have remained elusive for decades. Applying fundamental ecological principles to cancer biology models of metastasis provides novel insights into metastatic organotropism. We use critical concepts from foraging theory and movement ecology to propose that observed bone-specific metastasis is the result of habitat selection by foraging cancer cells. Furthermore, we posit that cancer cells can only perform habitat selection if and when they employ a reversible motile foraging strategy. Only a very small percentage of cells in a primary tumor harbor this ability. Therefore, our habitat selection model emphasizes the importance of identifying the rare subset of cancer cells that might exhibit habitat selection, ergo achieve bone-specific metastatic colonization.

Lamin A/C Mechanosensor Drives Tumor Cell Aggressiveness and Adhesion on Substrates With Tissue-Specific Elasticity.

Besides its structural properties in the nucleoskeleton, Lamin A/C is a mechanosensor protein involved in perceiving the elasticity of the extracellular matrix. In this study we provide evidence about Lamin A/C-mediated regulation of osteosarcoma cell adhesion and spreading on substrates with tissue-specific elasticities. Our working hypothesis is based on the observation that low-aggressive and bone-resident SaOS-2 osteosarcoma cells express high level of Lamin A/C in comparison to highly metastatic, preferentially to the lung, osteosarcoma 143B cells, thereby suggesting a role for Lamin A/C in tumor cell tropism. Specifically, LMNA gene over-expression in 143B cells induced a reduction in tumor cell aggressiveness in comparison to parental cells, with decreased proliferation rate and reduced migration capability. Furthermore, LMNA reintegration into 143B cells changed the adhesion properties of tumor cells, from a preferential tropism toward the 1.5 kPa PDMS substrate (resembling normal lung parenchyma) to the 28 kPa (resembling pre-mineralized bone osteoid matrix). Our study suggests that Lamin A/C expression could be involved in the organ tropism of tumor cells, thereby providing a rationale for further studies focused on the definition of cancer mechanism of metastatization.

Transcriptome analysis of heterogeneity in mouse model of metastatic breast cancer.

BACKGROUND: Cancer metastasis is a complex process involving the spread of malignant cells from a primary tumor to distal organs. Understanding this cascade at a mechanistic level could provide critical new insights into the disease and potentially reveal new avenues for treatment. Transcriptome profiling of spontaneous cancer models is an attractive method to examine the dynamic changes accompanying tumor cell spread. However, such studies are complicated by the underlying heterogeneity of the cell types involved. The purpose of this study was to examine the transcriptomes of metastatic breast cancer cells using the well-established MMTV-PyMT mouse model. METHODS: Organ-derived metastatic cell lines were harvested from 10 female MMTV-PyMT mice. Cancer cells were isolated and sorted based on the expression of CD44low/EpCAMhigh or CD44high/EpCAMhigh surface markers. RNA from each cell line was extracted and sequenced using the NextSeq 500 Illumina platform. Tissue-specific genes were compared across the different metastatic and primary tumor samples. Reads were mapped to the mouse genome using STAR, and gene expression was quantified using RSEM. Single-cell RNA-seq (scRNA-seq) was performed on select samples using the ddSeq platform by BioRad and analyzed using Seurat v3.2.3. Monocle2 was used to infer pseudo-time progression. RESULTS: Comparison of RNA sequencing data across all cell populations produced distinct gene clusters. Differential gene expression patterns related to CD44 expression, organ tropism, and immunomodulatory signatures were observed. scRNA-seq identified expression profiles based on tissue-dependent niches and clonal heterogeneity. These cohorts of data were narrowed down to identify subsets of genes with high expression and known metastatic propensity. Dot plot analyses further revealed clusters expressing cancer stem cell and cancer dormancy markers. Changes in relevant genes were investigated across pseudo-time and tissue origin using Monocle2. These data revealed transcriptomes that may contribute to sub-clonal evolution and treatment evasion during cancer progression. CONCLUSIONS: We performed a comprehensive transcriptome analysis of tumor heterogeneity and organ tropism during breast cancer metastasis. These data add to our understanding of metastatic progression and highlight targets for breast cancer treatment. These markers could also be used to image the impact of tumor heterogeneity on metastases.

Multiorgan tropism of SARS-CoV-2 lineage B.1.1.7.

Due to the development of novel functionalities, distinct SARS-CoV-2 variants such as B.1.1.7 fuel the current pandemic. B.1.1.7 is not only more transmissible, but may also cause an increased mortality compared to previous SARS-CoV-2 variants. Human tissue analysis of the SARS-CoV-2 lineage B.1.1.7 is urgently needed, and we here present autopsy data from 7 consecutive SARS-CoV-2 B.1.1.7 cases. The initial RT-qPCR analyses from nasopharyngeal swabs taken post mortem included typing assays for B.1.1.7. We quantitated SARS-CoV-2 B.1.1.7 viral load in autopsy tissue of multiple organs. Highest levels of SARS-CoV-2 B.1.1.7 copies normalized to ß-globin were detected in the respiratory system (lung and pharynx), followed by the liver and heart. Importantly, SARS-CoV-2 lineage B.1.1.7 was found in 100% of cases in the lungs and in 85.7% in pharynx tissue. Detection also in the kidney and brain highlighting a pronounced organ tropism. Comparison of the given results to a former cohort of SARS-CoV-2 deaths during the first wave in spring 2020 showed resembling organ tropism. Our results indicate that also SARS-CoV-2 B.1.1.7 has a relevant organ tropism beyond the respiratory tract. We speculate that B.1.1.7 spike protein's affinity to human ACE2 facilitates transmission, organ tropism, and ultimately morbidity and mortality. Further studies and larger cohorts are obligatory to proof this link.

The bone microenvironment invigorates metastatic seeds for further dissemination.

Metastasis has been considered as the terminal step of tumor progression. However, recent genomic studies suggest that many metastases are initiated by further spread of other metastases. Nevertheless, the corresponding pre-clinical models are lacking, and underlying mechanisms are elusive. Using several approaches, including parabiosis and an evolving barcode system, we demonstrated that the bone microenvironment facilitates breast and prostate cancer cells to further metastasize and establish multi-organ secondary metastases. We uncovered that this metastasis-promoting effect is driven by epigenetic reprogramming that confers stem cell-like properties on cancer cells disseminated from bone lesions. Furthermore, we discovered that enhanced EZH2 activity mediates the increased stemness and metastasis capacity. The same findings also apply to single cell-derived populations, indicating mechanisms distinct from clonal selection. Taken together, our work revealed an unappreciated role of the bone microenvironment in metastasis evolution and elucidated an epigenomic reprogramming process driving terminal-stage, multi-organ metastases.

Knockdown of AKT3 Activates HER2 and DDR Kinases in Bone-Seeking Breast Cancer Cells, Promotes Metastasis In Vivo and Attenuates the TGFß/CTGF Axis.

Bone metastases frequently occur in breast cancer patients and lack appropriate treatment options. Hence, understanding the molecular mechanisms involved in the multistep process of breast cancer bone metastasis and tumor-induced osteolysis is of paramount interest. The serine/threonine kinase AKT plays a crucial role in breast cancer bone metastasis but the effect of individual AKT isoforms remains unclear. Therefore, AKT isoform-specific knockdowns were generated on the bone-seeking MDA-MB-231 BO subline and the effect on proliferation, migration, invasion, and chemotaxis was analyzed by live-cell imaging. Kinome profiling and Western blot analysis of the TGFß/CTGF axis were conducted and metastasis was evaluated by intracardiac inoculation of tumor cells into NOD scid gamma (NSG) mice. MDA-MB-231 BO cells exhibited an elevated AKT3 kinase activity in vitro and responded to combined treatment with AKT- and mTOR-inhibitors. Knockdown of AKT3 significantly increased migration, invasion, and chemotaxis in vitro and metastasis to bone but did not significantly enhance osteolysis. Furthermore, knockdown of AKT3 increased the activity and phosphorylation of pro-metastatic HER2 and DDR1/2 but lowered protein levels of CTGF after TGFß-stimulation, an axis involved in tumor-induced osteolysis. We demonstrated that AKT3 plays a crucial role in bone-seeking breast cancer cells by promoting metastatic potential without facilitating tumor-induced osteolysis.

Organ tropism in solid tumor metastasis: an updated review.

Tumors are equipped with a highly complex machinery of interrelated events so as to adapt to hazardous conditions, preserve a growing cell mass and thrive at the site of metastasis. Tumor cells display metastatic propensity toward specific organs where the stromal milieu is appropriate for their further colonization. Effective colonization relies on the plasticity of tumor cells in adapting to the conditions of the new area by reshaping their epigenetic landscape. Breast cancer cells, for instance, are able to adopt brain-like or epithelial/osteoid features in order to pursue effective metastasis into brain and bone, respectively. The aim of this review is to discuss recent insights into organ tropism in tumor metastasis, outlining potential strategies to address this driver of tumor aggressiveness.

Triple-Negative Primary Breast Tumors Induce Supportive Premetastatic Changes in the Extracellular Matrix and Soluble Components of the Lung Microenvironment.

The lung is one of the deadliest sites of breast cancer metastasis, particularly in patients with triple-negative (TN) disease. We hypothesized that the presence of a TN primary breast tumor induces changes in the extracellular matrix (ECM) and soluble components of the lung microenvironment that support metastatic behavior. SUM159 (TN) and MCF7 (luminal A) breast cancer cells were injected into mice, and primary breast tumors were established prior to assessing metastatic niche changes. We observed increased CD117+ hematopoietic progenitor cells in the bone marrow of SUM159 mice versus MCF7 or control mice (p < 0.05). Relative to mice bearing MCF7 tumors and non-tumor controls, mice bearing SUM159 tumors demonstrated enhanced expression of ECM proteins in the lung (fibronectin, tenascin-c and periostin), with similar changes observed in lung fibroblasts treated with extracellular vesicles (EVs) from TN breast cancer cells (p < 0.05). Exposure to lung-conditioned media (LCM) from SUM159 tumor-bearing mice resulted in increased migration/proliferation of both SUM159 and MCF7 cells relative to the control (p < 0.05). In contrast, LCM from MCF-7 tumor-bearing mice had no such effect. LCM from SUM159 tumor-bearing mice contained 16 unique proteins relative to other LCM conditions, including the metastasis-associated proteins CCL7, FGFR4, GM-CSF, MMP3, thrombospondin-1 and VEGF. These findings suggest for the first time that the TN breast cancer molecular subtype may be an important determinant of premetastatic changes to both the ECM and soluble components of the lung, potentially mediated via breast cancer-derived EVs.

The Multiple Roles of Exosomes in Metastasis.

Exosomes are important contributors to cell-cell communication and their role as diagnostic markers for cancer and the pathogenesis for cancer is under intensive investigation. Here, we focus on their role in metastasis-related processes. We discuss their impact regarding promotion of invasion and migration of tumor cells, conditioning of lymph nodes, generation of premetastatic niches and organotropism of metastasis. Furthermore, we highlight interactions of exosomes with bone marrow and stromal components such as fibroblasts, endothelial cells, myeloid- and other immune-related cells in the context of metastases. For all processes as described above, we outline molecular and cellular components for therapeutic intervention with metastatic processes.