Building consensus on the application of organoid-based drug sensitivity testing in cancer precision medicine and drug development.

Patient-derived organoids (PDOs) have emerged as a promising platform for clinical and translational studies. A strong correlation exists between clinical outcomes and the use of PDOs to predict the efficacy of chemotherapy and/or radiotherapy. To standardize interpretation and enhance scientific communication in the field of cancer precision medicine, we revisit the concept of PDO-based drug sensitivity testing (DST). We present an expert consensus-driven approach for medication selection aimed at predicting patient responses. To further standardize PDO-based DST, we propose guidelines for clarification and characterization. Additionally, we identify several major challenges in clinical prediction when utilizing PDOs.

Hepatocyte-specific METTL3 ablation by Alb-iCre mice (GPT), but not by Alb-Cre mice (JAX), resulted in acute liver failure (ALF) and postnatal lethality.

AIM: In 2019, to examine the functions of METTL3 in liver and underlying mechanisms, we generated mice with hepatocyte-specific METTL3 homozygous knockout (METTL3Δhep) by simultaneously crossing METTL3fl/fl mice with Alb-iCre mice (GPT) or Alb-Cre mice (JAX), respectively. In this study, we explored the potential reasons why hepatocyte-specific METTL3 homozygous disruption by Alb-iCre mice (GPT), but not by Alb-Cre mice (JAX), resulted in acute liver failure (ALF) and then postnatal lethality. MAIN METHODS: Mice with hepatocyte-specific METTL3 knockout were generated by simultaneously crossing METTL3fl/fl mice with Alb-iCre mice (GPT; Strain No. T003814) purchased from the GemPharmatech Co., Ltd., (Nanjing, China) or with Alb-Cre mice (JAX; Strain No. 003574) obtained from The Jackson Laboratory, followed by combined-phenotype analysis. The publicly available RNA-sequencing data deposited in the NCBI Gene Expression Omnibus (GEO) database under the accession No.: GSE198512 (postnatal lethality), GSE197800 (postnatal survival) and GSE176113 (postnatal survival) were mined to explore the potential reasons why hepatocyte-specific METTL3 homozygous deletion by Alb-iCre mice (GPT), but not by Alb-Cre mice (JAX), leads to ALF and then postnatal lethality. KEY FINDINGS: Firstly, we observed that hepatocyte-specific METTL3 homozygous deficiency by Alb-iCre mice (GPT) or by Alb-Cre mice (JAX) caused liver injury, abnormal lipid accumulation and apoptosis. Secondly, we are surprised to find that hepatocyte-specific METTL3 homozygous deletion by Alb-iCre mice (GPT), but not by Alb-Cre mice (JAX), led to ALF and then postnatal lethality. Our findings clearly demonstrated that METTL3Δhep mice (GPT), which are about to die, exhibited the severe destruction of liver histological structure, suggesting that METTL3Δhep mice (GPT) nearly lose normal liver function, which subsequently contributes to ALF, followed by postnatal lethality. Finally, we unexpectedly found that as the compensatory growth responses of hepatocytes to liver injury induced by METTL3Δhep (GPT), the proliferation of METTL3Δhep hepatocytes (GPT), unlike METTL3Δhep hepatocytes (JAX), was not evidenced by the significant increase of Ki67-positive hepatocytes, not accompanied by upregulation of cell-cycle-related genes. Moreover, GO analysis revealed that upregulated genes in METTL3Δhep livers (GPT), unlike METTL3Δhep livers (JAX), are not functionally enriched in terms associated with cell cycle, cell division, mitosis, microtubule cytoskeleton organization, spindle organization, chromatin segregation and organization, and nuclear division, consistent with the loss of compensatory proliferation of METTL3Δhep hepatocytes (GPT) observed in vivo. Thus, obviously, the loss of the compensatory growth capacity of METTL3Δhep hepatocytes (GPT) in response to liver injury might contribute to, at least partially, ALF and subsequently postnatal lethality of METTL3Δhep mice (GPT). SIGNIFICANCE: These findings from this study and other labs provide strong evidence that these phenotypes (i.e., ALF and postnatal lethality) of METTL3Δhep mice (GPT) might be not the real functions of METTL3, and closely related with Alb-iCre mice (GPT), suggesting that we should remind researchers to use Alb-iCre mice (GPT) with caution to knockout gene in hepatocytes in vivo.

Predictive value of positive lymph node ratio in patients with locally advanced gastric remnant cancer.

BACKGROUND: Traditional lymph node stage (N stage) has limitations in advanced gastric remnant cancer (GRC) patients; therefore, establishing a new predictive stage is necessary. AIM: To explore the predictive value of positive lymph node ratio (LNR) according to clinicopathological characteristics and prognosis of locally advanced GRC. METHODS: Seventy-four patients who underwent radical gastrectomy and lymphadenectomy for locally advanced GRC were retrospectively reviewed. The relationship between LNR and clinicopathological characteristics was analyzed. The survival analysis was performed using Kaplan-Meier survival curves and Cox regression model. RESULTS: Number of metastatic LNs, tumor diameter, depth of tumor invasion, Borrmann type, serum tumor biomarkers, and tumor-node-metastasis (TNM) stage were correlated with LNR stage and N stage. Univariate analysis revealed that the factors affecting survival included tumor diameter, anemia, serum tumor biomarkers, vascular or neural invasion, combined resection, LNR stage, N stage, and TNM stage (all P < 0.05). The median survival time for those with LNR0, LNR1, LNR2 and LNR3 stage were 61, 31, 23 and 17 mo, respectively, and the differences were significant (P = 0.000). Anemia, tumor biomarkers and LNR stage were independent prognostic factors for survival in multivariable analysis (all P < 0.05). CONCLUSION: The new LNR stage is uniquely based on number of metastatic LNs, with significant prognostic value for locally advanced GRC, and could better differentiate overall survival, compared with N stage.

ThermomiR-377-3p-induced suppression of Cirbp expression is required for effective elimination of cancer cells and cancer stem-like cells by hyperthermia.

BACKGROUND: In recent years, the development of adjunctive therapeutic hyperthermia for cancer therapy has received considerable attention. However, the mechanisms underlying hyperthermia resistance are still poorly understood. In this study, we investigated the roles of cold­inducible RNA binding protein (Cirbp) in regulating hyperthermia resistance and underlying mechanisms in nasopharyngeal carcinoma (NPC). METHODS: CCK-8 assay, colony formation assay, tumor sphere formation assay, qRT-PCR, Western blot were employed to examine the effects of hyperthermia (HT), HT + oridonin(Ori) or HT + radiotherapy (RT) on the proliferation and stemness of NPC cells. RNA sequencing was applied to gain differentially expressed genes upon hyperthermia. Gain-of-function and loss-of-function experiments were used to evaluate the effects of RNAi-mediated Cirbp silencing or Cirbp overexpression on the sensitivity or resistance of NPC cells and cancer stem-like cells to hyperthermia by CCK-8 assay, colony formation assay, tumorsphere formation assay and apoptosis assay, and in subcutaneous xenograft animal model. miRNA transient transfection and luciferase reporter assay were used to demonstrate that Cirbp is a direct target of miR-377-3p. The phosphorylation levels of key members in ATM-Chk2 and ATR-Chk1 pathways were detected by Western blot. RESULTS: Our results firstly revealed that hyperthermia significantly attenuated the stemness of NPC cells, while combination treatment of hyperthermia and oridonin dramatically increased the killing effect on NPC cells and cancer stem cell (CSC)­like population. Moreover, hyperthermia substantially improved the sensitivity of radiation­resistant NPC cells and CSC­like cells to radiotherapy. Hyperthermia noticeably suppressed Cirbp expression in NPC cells and xenograft tumor tissues. Furthermore, Cirbp inhibition remarkably boosted anti­tumor­killing activity of hyperthermia against NPC cells and CSC­like cells, whereas ectopic expression of Cirbp compromised tumor­killing effect of hyperthermia on these cells, indicating that Cirbp overexpression induces hyperthermia resistance. ThermomiR-377-3p improved the sensitivity of NPC cells and CSC­like cells to hyperthermia in vitro by directly suppressing Cirbp expression. More importantly, our results displayed the significantly boosted sensitization of tumor xenografts to hyperthermia by Cirbp silencing in vivo, but ectopic expression of Cirbp almost completely counteracted hyperthermia-mediated tumor cell-killing effect against tumor xenografts in vivo. Mechanistically, Cirbp silencing-induced inhibition of DNA damage repair by inactivating ATM-Chk2 and ATR-Chk1 pathways, decrease in stemness and increase in cell death contributed to hyperthermic sensitization; conversely, Cirbp overexpression-induced promotion of DNA damage repair, increase in stemness and decrease in cell apoptosis contributed to hyperthermia resistance. CONCLUSION: Taken together, these findings reveal a previously unrecognized role for Cirbp in positively regulating hyperthermia resistance and suggest that thermomiR-377-3p and its target gene Cirbp represent promising targets for therapeutic hyperthermia.

Gut microbiome for predicting immune checkpoint blockade-associated adverse events.

BACKGROUND: The impact of the gut microbiome on the initiation and intensity of immune-related adverse events (irAEs) prompted by immune checkpoint inhibitors (ICIs) is widely acknowledged. Nevertheless, there is inconsistency in the gut microbial associations with irAEs reported across various studies. METHODS: We performed a comprehensive analysis leveraging a dataset that included published microbiome data (n = 317) and in-house generated data from 16S rRNA and shotgun metagenome samples of irAEs (n = 115). We utilized a machine learning-based approach, specifically the Random Forest (RF) algorithm, to construct a microbiome-based classifier capable of distinguishing between non-irAEs and irAEs. Additionally, we conducted a comprehensive analysis, integrating transcriptome and metagenome profiling, to explore potential underlying mechanisms. RESULTS: We identified specific microbial species capable of distinguishing between patients experiencing irAEs and non-irAEs. The RF classifier, developed using 14 microbial features, demonstrated robust discriminatory power between non-irAEs and irAEs (AUC = 0.88). Moreover, the predictive score from our classifier exhibited significant discriminative capability for identifying non-irAEs in two independent cohorts. Our functional analysis revealed that the altered microbiome in non-irAEs was characterized by an increased menaquinone biosynthesis, accompanied by elevated expression of rate-limiting enzymes menH and menC. Targeted metabolomics analysis further highlighted a notably higher abundance of menaquinone in the serum of patients who did not develop irAEs compared to the irAEs group. CONCLUSIONS: Our study underscores the potential of microbial biomarkers for predicting the onset of irAEs and highlights menaquinone, a metabolite derived from the microbiome community, as a possible selective therapeutic agent for modulating the occurrence of irAEs.

A loss-of-function mutation in human Oxidation Resistance 1 disrupts the spatial-temporal regulation of histone arginine methylation in neurodevelopment.

BACKGROUND: Oxidation Resistance 1 (OXR1) gene is a highly conserved gene of the TLDc domain-containing family. OXR1 is involved in fundamental biological and cellular processes, including DNA damage response, antioxidant pathways, cell cycle, neuronal protection, and arginine methylation. In 2019, five patients from three families carrying four biallelic loss-of-function variants in OXR1 were reported to be associated with cerebellar atrophy. However, the impact of OXR1 on cellular functions and molecular mechanisms in the human brain is largely unknown. Notably, no human disease models are available to explore the pathological impact of OXR1 deficiency. RESULTS: We report a novel loss-of-function mutation in the TLDc domain of the human OXR1 gene, resulting in early-onset epilepsy, developmental delay, cognitive disabilities, and cerebellar atrophy. Patient lymphoblasts show impaired cell survival, proliferation, and hypersensitivity to oxidative stress. These phenotypes are rescued by TLDc domain replacement. We generate patient-derived induced pluripotent stem cells (iPSCs) revealing impaired neural differentiation along with dysregulation of genes essential for neurodevelopment. We identify that OXR1 influences histone arginine methylation by activating protein arginine methyltransferases (PRMTs), suggesting OXR1-dependent mechanisms regulating gene expression during neurodevelopment. We model the function of OXR1 in early human brain development using patient-derived brain organoids revealing that OXR1 contributes to the spatial-temporal regulation of histone arginine methylation in specific brain regions. CONCLUSIONS: This study provides new insights into pathological features and molecular underpinnings associated with OXR1 deficiency in patients.

Faecalibacterium prausnitzii Abrogates Intestinal Toxicity and Promotes Tumor Immunity to Increase the Efficacy of Dual CTLA4 and PD-1 Checkpoint Blockade.

Immune checkpoint inhibitors (ICI) have revolutionized cancer therapy; however, their application is limited by the occurrence of immune-related adverse events. The gut microbiota plays important roles in the response to and toxicity of immunotherapy and Faecalibacterium prausnitzii (F. prausnitzii) has been shown to possess immunomodulatory potential. Here, we found that patients receiving ICIs who developed colitis had a lower abundance of F. prausnitzii. In vivo, immunocompetent mice administered with dextran sodium sulfate and immunodeficient NSG mice with human peripheral blood mononuclear cell transfer were treated with ICIs to study ICI-induced colitis. Dual CTLA4 and PD-1 blockade exacerbated autoimmune colitis, activated an inflammatory response, and promoted myeloid cell infiltration, with higher percentages of macrophages, dendritic cells, monocytes, and neutrophils. F. prausnitzii administration mitigated the exacerbated colitis induced by ICIs. Concomitantly, F. prausnitzii enhanced the antitumor immunity elicited by ICIs in tumor-bearing mice while abrogating colitis. In addition, administration of F. prausnitzii increased gut microbial alpha diversity and modulated the microbial composition, increasing a subset of gut probiotics and decreasing potential gut pathogens. F. prausnitzii abundance was reduced in mice that developed ICI-associated colitis. Together, this study shows that F. prausnitzii administration ameliorates ICI-induced colitis, reshapes the gut microbial composition, and enhances the antitumor activity of immunotherapy. SIGNIFICANCE: F. prausnitzii alleviates colitis while enhancing the tumor-suppressive effects of immune checkpoint blockade, indicating that supplementation with F. prausnitzii could be a treatment strategy to mitigate immunotherapy toxicity in patients with cancer.

Mettl3-mediated m6A modification plays a role in lipid metabolism disorders and progressive liver damage in mice by regulating lipid metabolism-related gene expression.

AIMS: N6-methyladenosine (m6A), the most abundant and conserved epigenetic modification of mRNA, participates in various physiological and pathological processes. However, the roles of m6A modification in liver lipid metabolism have yet to be understood entirely. We aimed to investigate the roles of the m6A "writer" protein methyltransferase-like 3 (Mettl3) in liver lipid metabolism and the underlying mechanisms. MAIN METHODS: We assessed the expression of Mettl3 in liver tissues of diabetes (db/db) mice, obese (ob/ob) mice, high saturated fat-, cholesterol-, and fructose-induced non-alcoholic fatty liver disease (NAFLD) mice, and alcohol abuse and alcoholism (NIAAA) mice by quantitative reverse-transcriptase PCR (qRT-PCR). Hepatocyte-specific Mettl3 knockout mice were used to evaluate the effects of Mettl3 deficiency in mouse liver. The molecular mechanisms underlying the roles of Mettl3 deletion in liver lipid metabolism were explored by multi-omics joint analysis of public data from the Gene Expression Omnibus database and further validated by qRT-PCR and Western blot. KEY FINDINGS: Significantly decreased Mettl3 expression was associated with NAFLD progression. Hepatocyte-specific knockout of Mettl3 resulted in significant lipid accumulation in the liver, increased serum total cholesterol levels, and progressive liver damage in mice. Mechanistically, loss of Mettl3 significantly downregulated the expression levels of multiple m6A-modified mRNAs related to lipid metabolism, including Adh7, Cpt1a, and Cyp7a1, further promoting lipid metabolism disorders and liver injury in mice. SIGNIFICANCE: In summary, our findings demonstrate that the expression alteration of genes related to lipid metabolism by Mettl3-mediated m6A modification contributes to the development of NAFLD.

Fusobacterium nucleatum-derived succinic acid induces tumor resistance to immunotherapy in colorectal cancer.

Immune checkpoint blockade therapy with anti-PD-1 monoclonal antibody (mAb) is a treatment for colorectal cancer (CRC). However, some patients remain unresponsive to PD-1 blockade. The gut microbiota has been linked to immunotherapy resistance through unclear mechanisms. We found that patients with metastatic CRC who fail to respond to immunotherapy had a greater abundance of Fusobacterium nucleatum and increased succinic acid. Fecal microbiota transfer from responders with low F. nucleatum, but not F. nucleatum-high non-responders, conferred sensitivity to anti-PD-1 mAb in mice. Mechanistically, F. nucleatum-derived succinic acid suppressed the cGAS-interferon-ß pathway, consequently dampening the antitumor response by limiting CD8+ T cell trafficking to the tumor microenvironment (TME) in vivo. Treatment with the antibiotic metronidazole reduced intestinal F. nucleatum abundance, thereby decreasing serum succinic acid levels and resensitizing tumors to immunotherapy in vivo. These findings indicate that F. nucleatum and succinic acid induce tumor resistance to immunotherapy, offering insights into microbiota-metabolite-immune crosstalk in CRC.

Hairy gene homolog increases nasopharyngeal carcinoma cell stemness by upregulating Bmi-1.

B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) is overexpressed in various cancer types. We found that Bmi-1 mRNA levels were elevated in nasopharyngeal carcinoma (NPC) cell lines. In immunohistochemical analyses, high Bmi-1 levels were observed in not only 5 of 38 non-cancerous nasopharyngeal squamous epithelial biopsies, but also in 66 of 98 NPC specimens (67.3%). High Bmi-1 levels were detected more frequently in T3-T4, N2-N3 and stage III-IV NPC biopsies than in T1-T2, N0-N1 and stage I-II NPC samples, indicating that Bmi-1 is upregulated in advanced NPC. In 5-8F and SUNE1 NPC cells, stable depletion of Bmi-1 using lentiviral RNA interference greatly suppressed cell proliferation, induced G1-phase cell cycle arrest, reduced cell stemness and suppressed cell migration and invasion. Likewise, knocking down Bmi-1 inhibited NPC cell growth in nude mice. Both chromatin immunoprecipitation and Western blotting assays demonstrated that Hairy gene homolog (HRY) upregulated Bmi-1 by binding to its promoter, thereby increasing the stemness of NPC cells. Immunohistochemistry and quantitative real-time PCR analyses revealed that HRY expression correlated positively with Bmi-1 expression in a cohort of NPC biopsies. These findings suggested that HRY promotes NPC cell stemness by upregulating Bmi-1, and that silencing Bmi-1 can suppress NPC progression.

MicroRNA-155 suppressed cholesterol-induced matrix degradation, pyroptosis and apoptosis by targeting RORα in nucleus pulposus cells.

Intervertebral disc degeneration (IDD) is associated with low back pain, yet its inherent mechanism remains obscure. Hypercholesteremia was regarded as a risk factor for IDD, and our previous study showed that cholesterol accumulation could elicit matrix degradation in the nucleus pulposus (NP). MicroRNA-155 (miR-155) was substantiated as protective in IDD, but its role in cholesterol-induced IDD was unclear. The present study investigated whether miR-155 could mediate cholesterol-related IDD and its internal mechanisms. In vivo experiments revealed high-fat diet-induced hypercholesteremia in wild-type (WT) mice along with the occurrence of IDD, whereas Rm155LG transgenic mice showed milder NP degeneration, as evidenced by Saffron O-fast green (SF) staining and immunohistochemistry (IHC). Meanwhile, IHC showed that NLRP3 and Bax expression was also suppressed in Rm155LG mice. In vitro studies using Western blotting (WB) and immunofluorescence (IF) confirmed that the miR-155 mimic could alleviate cholesterol-induced matrix degradation, apoptosis and pyroptosis in NP. Moreover, RORα was upregulated in severely degenerated NP compared to mild IDD. It was also noted that RORα was suppressed in Rm155LG mice. In this study, we demonstrated that miR-155 could target RORα and that inhibition of RORα could prevent cholesterol-induced matrix degradation, apoptosis, and pyroptosis in NP, indicating the protective effect of miR-155 in cholesterol-induced IDD by targeting RORα.

Mir155 regulates osteogenesis and bone mass phenotype via targeting S1pr1 gene.

MicroRNA-155 (miR155) is overexpressed in various inflammatory diseases and cancer, in which bone resorption and osteolysis are frequently observed. However, the role of miR155 on osteogenesis and bone mass phenotype is still unknown. Here, we report a low bone mass phenotype in the long bone of Mir155-Tg mice compared with wild-type mice. In contrast, Mir155-KO mice showed a high bone mass phenotype and protective effect against inflammation-induced bone loss. Mir155-KO mice showed robust bone regeneration in the ectopic and orthotopic model, but Mir155-Tg mice showed compromised bone regeneration compared with the wild-type mice. Similarly, the osteogenic differentiation potential of bone marrow stromal stem cells (BMSCs) from Mir155-KO mice was robust and Mir155-Tg was compromised compared with that of wild-type mice. Moreover, Mir155 knockdown in BMSCs from wild-type mice showed higher osteogenic differentiation potential, supporting the results from Mir155-KO mice. TargetScan analysis predicted sphingosine 1-phosphate receptor-1 (S1pr1) as a target gene of Mir155, which was further confirmed by luciferase assay and Mir155 knockdown. S1pr1 overexpression in BMSCs robustly promoted osteogenic differentiation without affecting cell viability and proliferation. Furthermore, osteoclastogenic differentiation of Mir155-Tg bone marrow-derived macrophages was inhibited compared with that of wild-type mice. Thus, Mir155 showed a catabolic effect on osteogenesis and bone mass phenotype via interaction with the S1pr1 gene, suggesting inhibition of Mir155 as a potential strategy for bone regeneration and bone defect healing.

Regulation mechanism of ferroptosis and its research progress in tumor immunotherapy.

Ferroptosis is a novel regulatory cell death, which is characterized by iron dependency and mainly caused by accumulation of intracellular lipid peroxides and reactive oxygen species. Ferroptosis plays an important role in the occurrence and development of a variety of malignant tumors, especially in anti-tumor treatment. As an emerging treatment method, the immunotherapy has been widely applied in the clinical practice, and the role of ferroptosis in tumor immunotherapy has been gradually explored. This study aims to illustrate the features of ferroptosis, and its role in anti-tumor immunotherapy and potential clinical application.

Autophagy induction promoted by m6A reader YTHDF3 through translation upregulation of FOXO3 mRNA.

Autophagy is crucial for maintaining cellular energy homeostasis and for cells to adapt to nutrient deficiency, and nutrient sensors regulating autophagy have been reported previously. However, the role of eiptranscriptomic modifications such as m6A in the regulation of starvation-induced autophagy is unclear. Here, we show that the m6A reader YTHDF3 is essential for autophagy induction. m6A modification is up-regulated to promote autophagosome formation and lysosomal degradation upon nutrient deficiency. METTL3 depletion leads to a loss of functional m6A modification and inhibits YTHDF3-mediated autophagy flux. YTHDF3 promotes autophagy by recognizing m6A modification sites around the stop codon of FOXO3 mRNA. YTHDF3 also recruits eIF3a and eIF4B to facilitate FOXO3 translation, subsequently initiating autophagy. Overall, our study demonstrates that the epitranscriptome regulator YTHDF3 functions as a nutrient responder, providing a glimpse into the post-transcriptional RNA modifications that regulate metabolic homeostasis.

Fluid nanoporous microinterface enables multiscale-enhanced affinity interaction for tumor-derived extracellular vesicle detection.

Tumor-derived extracellular vesicles (T-EVs) represent valuable markers for tumor diagnosis and treatment guidance. However, nanoscale sizes and the low abundance of marker proteins of T-EVs restrict interfacial affinity reaction, leading to low isolation efficiency and detection sensitivity. Here, we engineer a fluid nanoporous microinterface (FluidporeFace) in a microfluidic chip by decorating supported lipid bilayers (SLBs) on nanoporous herringbone microstructures with a multiscale-enhanced affinity reaction for efficient isolation of T-EVs. At the microscale level, the herringbone micropattern promotes the mass transfer of T-EVs to the surface. At the nanoscale level, nanoporousity can overcome boundary effects for close contact between T-EVs and the interface. At the molecular level, fluid SLBs afford clustering of recognition molecules at the binding site, enabling multivalent binding with an ∼83-fold increase of affinity compared with the nonfluid interface. With the synergetic enhanced mass transfer, interface contact, and binding affinity, FluidporeFace affords ultrasensitive detection of T-EVs with a limit of detection of 10 T-EVs µL-1, whose PD-L1 expression levels successfully distinguish cancer patients from healthy donors. We expect this multiscale enhanced interfacial reaction strategy will inspire the biosensor design and expand liquid biopsy applications, especially for low-abundant targets in clinical samples.

Colorectal cancer organoid models uncover oxaliplatin-resistant mechanisms at single cell resolution.

PURPOSE: Oxaliplatin-based chemotherapy is a standard treatment for advanced colorectal cancer (CRC) patients. However, chemoresistance-induced resistance is an essential cause for mortality. Therefore, it is necessary to study the mechanism of drug resistance in CRC. METHODS: Here, we established two strains of patient-derived organoids (PDOs) selected from oxaliplatin-resistant and treatment-naïve CRC patients. To dissect the drug-resistant mechanisms, these CRC-PDOs were subjected to single-cell RNA sequencing (scRNA-Seq). RESULTS: We found that the drug sensitivity test outcome from these organoids subjected to oxaliplatin and 5-FU exposure was consistent with the clinic readout. CRC-PDOs well recapitulated the morphology and histology of their parental biopsies based on HE and IHC staining of pathological biomarkers. The scRNA-Seq data filtered drug-resistant cell populations and related signaling pathways (e.g. oxidative phosphorylation and ATP metabolic process). The data also revealed several putative drug resistant-driven genes (STMN1, VEGFA and NDRG1) and transcription factors (E2F1, BRCA1, MYBL2, CDX2 and CDX1). CONCLUSION: We generated an oxaliplatin-resistant CRC organoid model that was employed to provide potential therapeutic targets for treating CRC patients exhibiting oxaliplatin-resistance.

Lactate receptor HCAR1 regulates neurogenesis and microglia activation after neonatal hypoxia-ischemia.

Neonatal cerebral hypoxia-ischemia (HI) is the leading cause of death and disability in newborns with the only current treatment being hypothermia. An increased understanding of the pathways that facilitate tissue repair after HI may aid the development of better treatments. Here, we study the role of lactate receptor HCAR1 in tissue repair after neonatal HI in mice. We show that HCAR1 knockout mice have reduced tissue regeneration compared with wildtype mice. Furthermore, proliferation of neural progenitor cells and glial cells, as well as microglial activation was impaired. Transcriptome analysis showed a strong transcriptional response to HI in the subventricular zone of wildtype mice involving about 7300 genes. In contrast, the HCAR1 knockout mice showed a modest response, involving about 750 genes. Notably, fundamental processes in tissue repair such as cell cycle and innate immunity were dysregulated in HCAR1 knockout. Our data suggest that HCAR1 is a key transcriptional regulator of pathways that promote tissue regeneration after HI.

Hypoxic-ischaemic brain injury is the most common cause of disability in newborn babies. This happens when the blood supply to the brain is temporarily blocked during birth and cells do not receive the oxygen and nutrients they need to survive. Cooling the babies down after the hypoxic-ischemic attack (via a technique called hypothermic treatment) can to some extent reduce the damage caused by the injury. However, doctors still need new drugs that can protect the brain and improve its recovery after the injury has occurred. Research in mice suggests that a chemical called lactate might help the brain to recover. Lactate is produced by muscles during hard exercise to provide energy to cells when oxygen levels are low. Recent studies have shown that it can also act as a signalling molecule that binds to a receptor called HCAR1 (short for hydroxycarboxylic acid receptor) on the surface of cells. However, it is poorly understood what role HCAR1 plays in the brain and whether it helps the brain recover from a hypoxic-ischaemic injury. To investigate, Kennedy et al. compared newborn mice with and without the gene that codes for HCAR1 that had undergone a hypoxic-ischaemic brain injury. While HCAR1 did not protect the mice from the disease, it did help their brains to heal. Mice with the gene for HCAR1 partly recovered some of their damaged brain tissue six weeks after the injury. Their cells switched on thousands of genes involved in the immune system and cell cycle, resulting in new brain cells being formed that could repopulate the injured areas. In contrast, the brain tissue of mice lacking HCAR1 barely produced any new cells. These findings suggest that HCAR1 may help with brain recovery after hypoxia-ischemia in newborn mice. This could lead to the development of drugs that might reduce or repair brain damage in newborn babies. However, further studies are needed to investigate whether HCAR1 has the same effect in humans.

A retrospective study of clinicopathological characteristics and prognostic factors of Krukenberg tumor with gastric origin.

Background: Krukenberg tumor (KT) of gastric origin has a poor prognosis. The present study of KTs are mainly case reports and clinical analysis with few samples. Therefore, it is urgent to explore the clinicopathologic characteristics of KTs through large sample studies. To improve the understanding of the clinical diagnosis and treatment of KT, this paper retrospectively analyzed 10 years of gastric cancer (GC) database data, including clinicopathological and prognostic features, aiming to provide a clinical reference for the diagnosis and treatment of the tumor. Methods: The clinicopathological characteristics, treatments, and survival data were collected and analyzed from 130 patients with KTs of GC. Clinicopathological data included clinical manifestations, laboratory findings, imaging reports, pathology and immunohistochemistry (IHC) reports. We collected treatment regimens information on whether they had undergone surgery and chemotherapy and performed survival follow-up. Univariate and multivariate analysis were used to investigate the risk factors of KTs with gastric origin. Results: The median age of the patients was 41 years. A total of 63.1% of patients had synchronous ovarian metastasis, 70.8% had bilateral ovarian metastasis, 68.5% had peritoneum metastasis, and 98.5% had pathologically poorly differentiated adenocarcinoma. The positive rate of human epidermal growth factor receptor 2 (HER-2) was 1.8%. The follow-up rate was 90.8%, and the median overall survival (mOS) of ovarian metastasis was 13.0 months. Univariate analysis showed statistically significant prognostic factors including menstrual status, size of the gastric lesions and ovarian metastases, number of lymph node metastasis, interval to ovarian metastasis, resection of gastric lesions, peritoneal metastasis, oophorectomy, chemotherapy after ovarian metastases, two-drug regimen chemotherapy, albumin, serum cancer antigen 125 (CA-125) levels, platelet count, D-dimer, fibrinogen, and high pretreatment platelet-to-lymphocyte ratio (PLR) and systemic immune-inflammation index (SII). Fibrinogen [hazard ration (HR) =0.483; 95% confidence interval (CI): 0.300-0.777; P=0.003], size of ovarian metastasis (HR =1.808; 95% CI: 1.178-2.776; P=0.007), chemotherapy after ovarian metastasis (HR =0.195; 95% CI: 0.101-0.379; P=0.000), peritoneal metastasis (HR =2.742; 95% CI: 1.606-4.682; P=0.000) and oophorectomy (HR =1.720; 95% CI: 1.066-2.778; P=0.026) were independent prognostic factors. Conclusions: GC patients with KTs have some unique clinical features. Hypercoagulable states, peritoneal metastasis, and untimely chemotherapy and oophorectomy might be a worse predictor for KTs derived from gastric origin.

A real-time pluripotency reporter for the long-term and real-time monitoring of pluripotency changes in induced pluripotent stem cells.

To master the technology of reprogramming mouse somatic cells to induced pluripotent stem cells (iPSCs), which will lay a good foundation for setting up a technology platform on reprogramming human cancer cells into iPSCs. Mouse iPSCs (i.e., Oct4-GFP miPSCs) was successfully generated from mouse embryonic fibroblasts (MEFs) harboring Oct4-EGFP transgene by introducing four factors, Oct4, Sox2, c-Myc and Klf4, under mESC (Murine embryonic stem cells) culture conditions. Oct4-GFP miPSCs were similar to mESCs in morphology, proliferation, mESC-specific surface antigens and gene expression. Additionally, Oct4-GFP miPSCs could be cultured in suspension to form embryoid bodies (EBs) and differentiate into cell types of the three germ layers in vitro. Moreover, Oct4-GFP miPSCs could develop to teratoma and chimera in vivo. Unlike cell cycle distribution of MEFs, Oct4-GFP miPSCs are similar to mESCs in the cell cycle structure which consists of higher S phase and lower G1 phase. More importantly, our data demonstrated that MEFs harboring Oct4-EGFP transgene did not express GFP, until they were reprogrammed to the pluripotent stage (iPSCs), while the GFP expression was progressively lost when these pluripotent Oct4-GFP miPSCs exposed to EB-mediated differentiation conditions, suggesting the pluripotency of Oct4-GFP miPSCs can be real-time monitored over long periods of time via GFP assay. Altogether, our findings demonstrate that Oct4-GFP miPSC line is successfully established, which will lay a solid foundation for setting up a technology platform on reprogramming cancer cells into iPSCs. Furthermore, this pluripotency reporter system permits the long-term real-time monitoring of pluripotency changes in a live single-cell, and its progeny.