Circulating tumor cell phenotype detection based on epithelial-mesenchymal transition markers combined with clinicopathological risk has potential to better predict recurrence in stage III breast cancer treated with neoadjuvant chemotherapy: a pilot study.

BACKGROUND: The potential value of detecting epithelial-mesenchymal transition (EMT) CTCs in early breast cancer, especially during the neoadjuvant therapy period, requires further investigation. We analyzed dynamic CTC phenotype status, to improve recurrence risk stratification for patients with stage III breast cancers. METHODS: We enrolled 45 patients with stage III breast cancers from 2 clinical trials undergoing neoadjuvant chemotherapy and utilized the CanPatrol CTC enrichment technique pre- and post-chemotherapy to identify CTC phenotypes, including epithelial CTCs, biphenotypic epithelial/mesenchymal CTCs, and mesenchymal CTCs, in peripheral blood samples. Kaplan-Meier analyses were conducted to explore the prognostic value of dynamic change of CTC count and the proportion of CTCs with different phenotypes. Then, redefine the risk stratification based on CTC status and clinicopathological risk in combination. RESULTS: Increased proportion of M + CTCs was a high-risk CTC status that was associated with decreased DFS (HR, 3.584; 95% CI, 1.057-12.15). In a combined analysis with clinicopathological risk, patients with high-risk tumors had an elevated risk of recurrence compared to patients with low-risk tumors (HR, 4.482; 95% CI, 1.246-16.12). The recurrence risk could be effectively stratified by newly defined risk stratification criteria, with 5-year DFS of 100.0%, 77.3%, and 50.0%, respectively, for low-risk, mid-risk, and high-risk patients (P = 0.0077). Finally, in the ROC analysis, the redefined risk stratification demonstrated higher predictive significance with an AUC of 0.7727, compared to CTC status alone (AUC of 0.6751) or clinicopathological risk alone (AUC of 0.6858). CONCLUSION: The proportion of M + CTCs increased after neoadjuvant chemotherapy indicating a higher risk of tumor recurrence. Combining CTC status with clinicopathological risk has potential to redefine the risk stratification of stage III breast cancers and provide improved predictions of relapse.

Tumor Dormancy and Reactivation: The Role of Heat Shock Proteins.

Tumors are a heterogeneous group of cell masses originating in various organs or tissues. The cellular composition of the tumor cell mass interacts in an intricate manner, influenced by humoral, genetic, molecular, and tumor microenvironment cues that dictate tumor growth or suppression. As a result, tumors undergo a period of a dormant state before their clinically discernible stage, which surpasses the clinical dormancy threshold. Moreover, as a genetically imprinted strategy, early-seeder cells, a distinct population of tumor cells, break off to dock nearby or extravasate into blood vessels to secondary tissues, where they form disseminated solitary dormant tumor cells with reversible capacity. Among the various mechanisms underlying the dormant tumor mass and dormant tumor cell formation, heat shock proteins (HSPs) might play one of the most important roles in how the dormancy program plays out. It is known that numerous aberrant cellular processes, such as malignant transformation, cancer cell stemness, tumor invasion, metastasis, angiogenesis, and signaling pathway maintenance, are influenced by the HSPs. An accumulating body of knowledge suggests that HSPs may be involved in the angiogenic switch, immune editing, and extracellular matrix (ECM) remodeling cascades, crucial genetically imprinted strategies important to the tumor dormancy initiation and dormancy maintenance program. In this review, we highlight the biological events that orchestrate the dormancy state and the body of work that has been conducted on the dynamics of HSPs in a tumor mass, as well as tumor cell dormancy and reactivation. Additionally, we propose a conceptual framework that could possibly underlie dormant tumor reactivation in metastatic relapse.

Hydrogel activation of Mincle receptors for tumor cell processing: A novel approach in cancer immunotherapy.

An obstacle in current tumor immunotherapies lies in the challenge of achieving sustained and tumor-targeting T cell immunity, impeded by the limited antigen processing and cross-presentation of tumor antigens. Here, we propose a hydrogel-based multicellular immune factory within the body that autonomously converts tumor cells into an antitumor vaccine. Within the body, the scaffold, formed by a calcium-containing chitosan hydrogel complex (ChitoCa) entraps tumor cells and attracts immune cells to establish a durable and multicellular microenvironment. Within this context, tumor cells are completely eliminated by antigen-presenting cells (APCs) and processed for cross-antigen presentation. The regulatory mechanism relies on the Mincle receptor, a cell-phagocytosis-inducing C-type lectin receptor specifically activated on ChitoCa-recruited APCs, which serves as a recognition synapse, facilitating a tenfold increase in tumor cell engulfment and subsequent elimination. The ChitoCa-induced tumor cell processing further promotes the cross-presentation of tumor antigens to prime protective CD8+ T cell responses. Therefore, the ChitoCa treatment establishes an immune niche within the tumor microenvironment, resulting in effective tumor regression either used alone or in combination with other immunotherapies. This hydrogel-induced immune factory establishes a functional organ-like multicellular colony for tumor-specific immunotherapy, paving the way for innovative strategies in cancer treatment.

Advances in the role of circulating tumor cell heterogeneity in metastatic small cell lung cancer.

Small cell lung cancer (SCLC), a highly aggressive malignancy, is rapidly at an extensive stage once diagnosed and is one of the leading causes of death from malignancy. In the past decade, the treatment of SCLC has largely remained unchanged, and chemotherapy remains the cornerstone of SCLC treatment. The therapeutic value of adding immune checkpoint inhibitors to chemotherapy for SCLC is low, and only a few SCLC patients have shown a response to immune checkpoint inhibitors. Circulating tumor cells (CTCs) are tumor cells shed from solid tumor masses into the peripheral circulation and are key to tumor metastasis. Single-cell sequencing has revealed that the genetic profiles of individual CTCs are highly heterogeneous and contribute to the poor outcome and prognosis of SCLC patients. Theoretically, phenotypic analysis of CTCs may be able to predict the diagnostic significance of new potential targets for metastatic tumors. In this paper, we will discuss in depth the heterogeneity of CTCs in SCLC and the value of CTCs for the diagnosis and prognosis of SCLC and as relevant tumor markers in metastatic SCLC.

3D engineered scaffold for large-scale Vigil immunotherapy production.

Previously, we reported successful cellular expansion of a murine colorectal carcinoma cell line (CT-26) using a three-dimensional (3D) engineered extracellular matrix (EECM) fibrillar scaffold structure. CCL-247 were grown over a limited time period of 8 days on 3D EECM or tissue culture polystyrene (TCPS). Cells were then assayed for growth, electroporation efficiency and Vigil manufacturing release criteria. Using EECM scaffolds, we report an expansion of CCL-247 (HCT116), a colorectal carcinoma cell line, from a starting concentration of 2.45 × 105 cells to 1.9 × 106 cells per scaffold. Following expansion, 3D EECM-derived cells were assessed based on clinical release criteria of the Vigil manufacturing process utilized for Phase IIb trial operation with the FDA. 3D EECM-derived cells passed all Vigil manufacturing release criteria including cytokine expression. Here, we demonstrate successful Vigil product manufacture achieving the specifications necessary for the clinical trial product release of Vigil treatment. Our results confirm that 3D EECM can be utilized for the expansion of human cancer cell CCL-247, justifying further clinical development involving human tissue sample manufacturing including core needle biopsy and minimal ascites samples.

Two-pronged anti-cancer nanovaccines enpowered by exogenous/endogenous tumor-associated antigens.

Cancer vaccines based on single-source (exogenous or endogenous) tumor-associated antigens (TAAs) are often challenged by the insufficient T cell response and the immunosuppressive tumor microenvironment (TME). Herein, a dual TAAs-boosted nanovaccine based on cancer cell (4T1) membrane-cloaked, CO-immobilized Prussian blue nanoparticles (4T1-PB-CO NPs) is developed and coupled with anti-interleukin (IL)-10 therapy to maximize the efficacy of antitumor immunotherapy. 4T1 cell membrane not only endows NPs with tumor targeting ability, but also serves as exogenous TAAs to trigger CD4+ T cell response and M1-phenotype polarization of tumor-associated macrophages. Under near-infrared light irradiation, 4T1-PB-CO NPs release CO to induce immunogenic cell death (ICD) of tumor cells, thus generating endogenous TAAs to activate CD8+ T cell response. Meanwhile, ICD triggers release of damage-associated molecular patterns, which could promote DC maturation to amplify the antitumor T cell response. When combined with anti-IL-10 that reverses the immunosuppressive TME, 4T1-PB-CO NPs efficiently suppress the primary tumors and produce an abscopal effect to inhibit distant tumors in a breast tumor-bearing mouse model. Such a two-pronged cancer vaccine represents a promising paradigm for robust antitumor immunotherapy.

Circulating tumor cells with increasing aneuploidy predict inferior prognosis and therapeutic resistance in small cell lung cancer.

AIMS: Treatment resistance commonly emerges in small cell lung cancer (SCLC), necessitating the development of novel and effective biomarkers to dynamically assess therapeutic efficacy. This study aims to evaluate the clinical utility of aneuploid circulating tumor cells (CTCs) for risk stratification and treatment response monitoring. METHODS: A total of 126 SCLC patients (two cohorts) from two independent cancer centers were recruited as the study subjects. Blood samples were collected from these patients and aneuploid CTCs were detected. Aneuploid CTC count (ACC) and aneuploid CTC score (ACS), were used to predict progression-free survival (PFS) and overall survival (OS). The performance of the ACC and the ACS was evaluated by calculating the area under the receiver operating characteristic (ROC) curve (AUC). RESULTS: Compared to ACC, ACS exhibited superior predictive power for PFS and OS in these 126 patients. Moreover, both univariate and multivariate analyses revealed that ACS was an independent prognostic factor. Dynamic ACS changes reflected treatment response, which is more precise than ACC changes. ACS can be used to assess chemotherapy resistance and is more sensitive than radiological examination (with a median lead time of 2.8 months; P < 0.001). When patients had high ACS levels (> 1.115) at baseline, the combination of immunotherapy and chemotherapy resulted in longer PFS (median PFS, 7.7 months; P = 0.007) and OS (median OS, 16.3 months; P = 0.033) than chemotherapy alone (median PFS, 4.9 months; median OS, 13.6 months). CONCLUSIONS: ACS could be used as a biomarker for risk stratification, treatment response monitoring, and individualized therapeutic intervention in SCLC patients.

Spatial distribution pattern of immune cells is associated with patient prognosis in colorectal cancer.

BACKGROUND: The spatial context of tumor-infiltrating immune cells (TIICs) is important in predicting colorectal cancer (CRC) patients' clinical outcomes. However, the prognostic value of the TIIC spatial distribution is unknown. Thus, we aimed to investigate the association between TIICs in situ and patient prognosis in a large CRC sample. METHODS: We implemented multiplex immunohistochemistry staining technology in 190 CRC samples to quantify 14 TIIC subgroups in situ. To delineate the spatial relationship of TIICs to tumor cells, tissue slides were segmented into tumor cell and microenvironment compartments based on image recognition technology, and the distance between immune and tumor cells was calculated by implementing the computational pipeline phenoptr. RESULTS: MPO+ neutrophils and CD68+IDO1+ tumor-associated macrophages (TAMs) were enriched in the epithelial compartment, and myeloid lineage cells were located nearest to tumor cells. Except for CD68+CD163+ TAMs, other cells were all positively associated with favorable prognosis. The prognostic predictive power of TIICs was highly related to their distance to tumor cells. Unsupervised clustering analysis divided colorectal cancer into three subtypes with distinct prognostic outcomes, and correlation analysis revealed the synergy among B cells, CD68+IDO1+TAMs, and T lineage cells in producing an effective immune response. CONCLUSIONS: Our study suggests that the integration of spatial localization with TIIC abundance is important for comprehensive prognostic assessment.

Establishment and characterization of a novel multidrug-resistant pancreatic ductal adenocarcinoma cell line, PDAC-X1.

Drug resistance remains a significant challenge in the treatment of pancreatic cancer. The development of drug-resistant cell lines is crucial to understanding the underlying mechanisms of resistance and developing novel drugs to improve clinical outcomes. Here, a novel pancreatic cancer cell line, PDAC-X1, derived from Chinese patients has been established. PDAC-X1 was characterized by the immune phenotype, biology, genetics, molecular characteristics, and tumorigenicity. In vitro analysis revealed that PDAC-X1 cells exhibited epithelial morphology and cell markers (CK7 and CK19), expressed cancer-associated markers (E-cadherin, Vimentin, Ki-67, CEA, CA19-9), and produced pancreatic cancer-like organs in suspension culture. In vivo analysis showed that PDAC-X1 cells maintained tumorigenicity with a 100% tumor formation rate. This cell line exhibited a complex karyotype, dominated by subtriploid karyotypes. In addition, PDAC-X1 cells exhibited intrinsic multidrug resistance to multiple drugs, including gemcitabine, paclitaxel, 5-fluorouracil, and oxaliplatin. In conclusion, the PDAC-X1 cell line has been established and characterized, representing a useful and valuable preclinical model to study the underlying mechanisms of drug resistance and develop novel drug therapeutics to improve patient outcomes.

Boron Neutron Capture Therapy-Derived Extracellular Vesicles via DNA Accumulation Boost Antitumor Dendritic Cell Vaccine Efficacy.

Radiated tumor cell-derived extracellular vesicles (RT-EVs) encapsulate abundant DNA fragments from irradiated tumor cells, in addition to acting as integrators of multiple tumor antigens. Accumulating evidence indicates these DNA fragments from damaged cells are involved in downstream immune responses, but most of them are degraded in cells before incorporation into derived RT-EVs, thus the low abundance of DNA fragments limits immune responses of RT-EVs. Here, this study found that different radiations affected fates of DNA fragments in RT-EVs. Boron neutron capture therapy (BNCT) induced DNA accumulation in RT-EVs (BEVs) by causing more DNA breaks and DNA oxidation resisting nuclease degradation. This is attributed to the high-linear energy transfer (LET) properties of alpha particles from the neutron capture reaction of 10B. When being internalized by dendritic cells (DCs), BEVs activated the DNA sensing pathway, resulting in functional enhancements including antigen presentation, migration capacity, and cytokine secretion. After vaccination of the BEVs-educated DCs (BEV@BMDCs), the effector T cells significantly expanded and infiltrated into tumors, suggesting robust anti-tumor immune activation. BEV@BMDCs not only effectively inhibited the primary tumor growth and metastasis formation but also elicited long-term immune memory. In conclusion, a successful DC vaccine is provided as a promising candidate for tumor vaccine.

Radiomics and visual analysis for predicting success of transplantation of heterotopic glioblastoma in mice with MRI.

BACKGROUND: Quantifying tumor growth and treatment response noninvasively poses a challenge to all experimental tumor models. The aim of our study was, to assess the value of quantitative and visual examination and radiomic feature analysis of high-resolution MR images of heterotopic glioblastoma xenografts in mice to determine tumor cell proliferation (TCP). METHODS: Human glioblastoma cells were injected subcutaneously into both flanks of immunodeficient mice and followed up on a 3 T MR scanner. Volumes and signal intensities were calculated. Visual assessment of the internal tumor structure was based on a scoring system. Radiomic feature analysis was performed using MaZda software. The results were correlated with histopathology and immunochemistry. RESULTS: 21 tumors in 14 animals were analyzed. The volumes of xenografts with high TCP (H-TCP) increased, whereas those with low TCP (L-TCP) or no TCP (N-TCP) continued to decrease over time (p < 0.05). A low intensity rim (rim sign) on unenhanced T1-weighted images provided the highest diagnostic accuracy at visual analysis for assessing H-TCP (p < 0.05). Applying radiomic feature analysis, wavelet transform parameters were best for distinguishing between H-TCP and L-TCP / N-TCP (p < 0.05). CONCLUSION: Visual and radiomic feature analysis of the internal structure of heterotopically implanted glioblastomas provide reproducible and quantifiable results to predict the success of transplantation.

Spontaneous-stimulated Raman co-localization dual-modal analysis approach for efficient identification of tumor cells.

The study of highly heterogeneous tumor cells, especially acute myeloid leukemia (AML) cells, usually relies on invasive analytical methods such as morphology, immunology, cytogenetics, and molecular biology classification, which are complex and time-consuming to perform. Mortality is high if patients are not diagnosed in a timely manner, so rapid label-free analysis of gene expression and metabolites within single-cell substructures is extremely important for clinical diagnosis and treatment. As a label-free and non-destructive vibrational detection technique, spontaneous Raman scattering provides molecular information across the full spectrum of the cell but lacks rapid imaging localization capabilities. In contrast, stimulated Raman scattering (SRS) provides a high-speed, high-resolution imaging view that can offer real-time subcellular localization assistance for spontaneous Raman spectroscopic detection. In this paper, we combined multi-color SRS microscopy with spontaneous Raman to develop a co-localized Raman imaging and spectral detection system (CRIS) for high-speed chemical imaging and quantitative spectral analysis of subcellular structures. Combined with multivariate statistical analysis methods, CRIS efficiently differentiated AML from normal leukocytes with an accuracy of 98.1 % and revealed the differences in the composition of nuclei and cytoplasm of AML relative to normal leukocytes. Compared to conventional Raman spectroscopy blind sampling without imaging localization, CRIS increased the efficiency of single-cell detection by at least three times. In addition, using the same approach for further identification of AML subtypes M2 and M3, we demonstrated that intracytoplasmic differential expression of proteins is a marker for their rapid and accurate classifying. CRIS analysis methods are expected to pave the way for clinical translation of rapid tumor cell identification.

Single-cell analyses reveal evolution mimicry during the specification of breast cancer subtype.

Background: The stem or progenitor antecedents confer developmental plasticity and unique cell identities to cancer cells via genetic and epigenetic programs. A comprehensive characterization and mapping of the cell-of-origin of breast cancer using novel technologies to unveil novel subtype-specific therapeutic targets is still absent. Methods: We integrated 195,144 high-quality cells from normal breast tissues and 406,501 high-quality cells from primary breast cancer samples to create a large-scale single-cell atlas of human normal and cancerous breasts. Potential heterogeneous origin of malignant cells was explored by contrasting cancer cells against reference normal epithelial cells. Multi-omics analyses and both in vitro and in vivo experiments were performed to screen and validate potential subtype-specific treatment targets. Novel biomarkers of identified immune and stromal cell subpopulations were validated by immunohistochemistry in our cohort. Results: Tumor stratification based on cancer cell-of-origin patterns correlated with clinical outcomes, genomic aberrations and diverse microenvironment constitutions. We found that the luminal progenitor (LP) subtype was robustly associated with poor prognosis, genomic instability and dysfunctional immune microenvironment. However, the LP subtype patients were sensitive to neoadjuvant chemotherapy (NAC), PARP inhibitors (PARPi) and immunotherapy. The LP subtype-specific target PLK1 was investigated by both in vitro and in vivo experiments. Besides, large-scale single-cell profiling of breast cancer inspired us to identify a range of clinically relevant immune and stromal cell subpopulations, including subsets of innate lymphoid cells (ILCs), macrophages and endothelial cells. Conclusion: The present single-cell study revealed the cellular repertoire and cell-of-origin patterns of breast cancer. Combining single-cell and bulk transcriptome data, we elucidated the evolution mimicry from normal to malignant subtypes and expounded the LP subtype with vital clinical implications. Novel immune and stromal cell subpopulations of breast cancer identified in our study could be potential therapeutic targets. Taken together, Our findings lay the foundation for the precise prognostic and therapeutic stratification of breast cancer.

Detection and comparison of tumor cell-associated microbiota from different compartments of colorectal cancer.

Introduction: Intratumoral microbes play an important role in the development of colorectal cancer (CRC). However, studying intratumoral microbes in CRC faces technical challenges, as tumor microbe communities are often contaminated by fecal microbes due to the structure of the gut folds and villi. The present study aimed to develop a new method for isolating tumor cell-associated microbiota and comparing microbial populations from different compartments. Materials and methods: The distribution of intestinal bacteria was detected using immunohistochemistry combined with 5R-16s rRNA gene sequencing to explore the effects of the sampling site and number of washes on the detection of microbiota. The 5R-16s rRNA gene sequencing was performed using 44 samples from 11 patients with CRC, including CRC tumor tissues (TT), normal tissues adjacent to CRC (NT), tumor cells (TC), and normal cells (NC). TC and NC were obtained from the TT and NT using an enzymatic digestion method. The microbiota and their potential functions in the four groups were analyzed and compared to determine the differential microbiota related to CRC. Results: Bacteria were mainly distributed in the feces covering intestinal tissues and in the epithelial cells and macrophages within the tissues. Different sampling sites and number of washes led to detection of different microbiota distributions. Although the cleaning method could be controlled, sampling sites varied and led to different microbiota distributions. The phyla of Firmicutes and Bacteroidetes were highly abundant in the conventionally used tissue samples, whereas Proteobacteria was the most abundant phyla in the cell samples isolated with the new method (i.e., after cell enzymatic hydrolysis). Detection of CRC cell-associated microbiota using a cell enzymatic digestion method showed that some bacteria, such as Fusobacterium, Eikenella, Shewanella, and Listeria, were more abundant in TT than NT, whereas the abundance of Akkermansia was lower in TT than NT. The tumor/normal ratios of some bacteria, such as Gemella, Escherichia, Shigella, and Blautia, were different between the cell and tissue samples. Conclusion: The cell enzymatic digestion method reduced fecal bacterial contamination, enabling low biomass intratumoral microbiota to be detected and allowing prediction of bacterial distributions.

Characterisation of circulating tumor-associated and immune cells in patients with advanced-stage non-small cell lung cancer.

Objectives: Globally, non-small cell lung cancer (NSCLC) is the most prevalent form of lung cancer and the leading cause of cancer-related deaths. Tumor-associated circulating cells in NSCLC can have a wide variety of morphological and phenotypic characteristics, including epithelial, immunological or hybrid subtypes. The distinctive characteristics and potential clinical significance of these cells in patients with NSCLC are explored in this study. Methods: We utilised a spiral microfluidic device to enrich large cells and cell aggregates from the peripheral blood samples of NSCLC patients. These cells were characterised through high-resolution immunofluorescent imaging and statistical analysis, correlating findings with clinical information from our patient cohort. Results: We have identified varied populations of heterotypic circulating tumor cell clusters with differing immune cell composition that included a distinct class of atypical tumor-associated macrophages that exhibits unique morphology and cell size. This subtype's prevalence is positively correlated with the tumor stage, progression and metastasis. Conclusions: Our study reveals a heterogeneous landscape of circulating tumor cells and their clusters, underscoring the complexity of NSCLC pathobiology. The identification of a unique subtype of atypical tumor-associatedmacrophages that simultaneously express both tumor and immune markers and whose presence correlates with late disease stages, poor clinical outcomes and metastatic risk infers  the potential of these cells as biomarkers for NSCLC staging and prognosis. Future studies should focus on the role of these cells in the tumor microenvironment and their potential as therapeutic targets. Additionally, longitudinal studies tracking these cell types through disease progression could provide further insights into their roles in NSCLC evolution and response to treatment.

Detection of PD­L1 expression and epithelial­mesenchymal transition of circulating tumor cells in non­small cell lung cancer.

The present study aimed to assess the roles of peripheral circulating tumor cell (CTC) count, CTC subtypes and programmed death ligand 1 (PD-L1) expression in the clinical staging and prognosis of patients with non-small cell lung cancer (NSCLC). A total of 100 patients with NSCLC with available tumor tissues were enrolled in the present study, and 7.5 ml peripheral blood was collected. Patients were divided into PD-L1-positive and PD-L1-negative groups according to PD-L1 immunohistochemical staining. Peripheral blood samples from both groups were analyzed to determine the CTC count, epithelial-type CTCs (E-CTCs), mesenchymal-type CTCs (M-CTCs) and PD-L1 expression. Clinical data were collected, and patients were followed up for a maximum of 36 months, with patient death as the endpoint event. Patients with PD-L1-positive tumors had a worse prognosis compared with those with PD-L1-negative tumors (P=0.045). The PD-L1-positive group exhibited significantly higher numbers of CTCs and M-CTCs compared with the PD-L1-negative group (P≤0.05). However, the number of E-CTCs did not differ significantly between the two groups (P>0.05). PD-L1-positive patients with higher CTC and M-CTC counts had relatively poorer prognoses (P≤0.05), while the number of E-CTCs had no significant effect on prognosis (P>0.05). Compared with the early-stage NSCLC group, the late-stage NSCLC group exhibited a significant increase in the CTC count (P≤0.05), while E-CTC and M-CTC counts did not significantly differ between the two groups (P>0.05). The PD-L1-positive group exhibited a significant increase in the number of PD-L1+ CTCs and PD-L1+ M-CTCs compared with the PD-L1-negative group (P≤0.05), while PD-L1+ E-CTC counts did not differ significantly between the two groups (P>0.05). The PD-L1-positive patients with a higher number of PD-L1+ CTCs and PD-L1+ M-CTCs had relatively poorer prognoses (P≤0.05), while the PD-L1+ E-CTC count had no significant effect on prognosis (P>0.05). Compared with the early-stage NSCLC group, the late-stage NSCLC group exhibited a significant increase in the number of PD-L1+ CTCs and PD-L1+ M-CTCs (P≤0.05), while PD-L1+ E-CTC counts did not significantly differ between the two groups (P>0.05). Based on univariate and multivariate analyses, the number of PD-L1+ M-CTCs was identified as an independent prognostic factor for NSCLC. In conclusion, the presence of CTCs in peripheral blood, particularly PD-L1+ M-CTC subtype, indicated poorer clinical staging and prognosis in patients with NSCLC. These findings suggested that CTCs, specifically the PD-L1+ M-CTC subtype, could serve as a monitoring indicator for the clinical staging and prognosis of patients with NSCLC.

Dual-mRNA Delivery Using Tumor Cell Lysate-Based Multifunctional Nanoparticles as an Efficient Colon Cancer Immunogene Therapy.

Background: Messenger RNA (mRNA)-based immunogene therapy holds significant promise as an emerging tumor therapy approach. However, the delivery efficiency of existing mRNA methods and their effectiveness in stimulating anti-tumor immune responses require further enhancement. Tumor cell lysates containing tumor-specific antigens and biomarkers can trigger a stronger immune response to tumors. In addition, strategies involving multiple gene therapies offer potential optimization paths for tumor gene treatments. Methods: Based on the previously developed ideal mRNA delivery system called DOTAP-mPEG-PCL (DMP), which was formed through the self-assembly of 1.2-dioleoyl-3-trimethylammonium-propane (DOTAP) and methoxypoly (ethylene glycol)-b-poly (ε-caprolactone) (mPEG-PCL), we introduced a fused cell-penetrating peptide (fCPP) into the framework and encapsulated tumor cell lysates to form a novel nanovector, termed CLSV system (CLS: CT26 tumor cell lysate, V: nanovector). This system served a dual purpose of facilitating the delivery of two mRNAs and enhancing tumor immunogene therapy through tumor cell lysates. Results: The synthesized CLSV system had an average size of 241.17 nm and a potential of 39.53 mV. The CLSV system could not only encapsulate tumor cell lysates, but also deliver two mRNAs to tumor cells simultaneously, with a transfection efficiency of up to 60%. The CLSV system effectively activated the immune system such as dendritic cells to mature and activate, leading to an anti-tumor immune response. By loading Bim-encoded mRNA and IL-23A-encoded mRNA, CLSV/Bim and CLSV/IL-23A complexes were formed, respectively, to further induce apoptosis and anti-tumor immunity. The prepared CLSV/dual-mRNA complex showed significant anti-cancer effects in multiple CT26 mouse models. Conclusion: Our results suggest that the prepared CLSV system is an ideal delivery system for dual-mRNA immunogene therapy.

Tethering of hexokinase 2 to mitochondria promotes resistance of liver cancer cells to natural killer cell cytotoxicity.

Hexokinases (HKs) control the first step of glucose catabolism. A switch of expression from liver HK (glucokinase, GCK) to the tumor isoenzyme HK2 is observed in hepatocellular carcinoma progression. Our prior work revealed that HK isoenzyme switch in hepatocytes not only regulates hepatic metabolic functions but also modulates innate immunity and sensitivity to Natural Killer (NK) cell cytotoxicity. This study investigates the impact of HK2 expression and its mitochondrial binding on the resistance of human liver cancer cells to NK-cell-induced cytolysis. We have shown that HK2 expression induces resistance to NK cell cytotoxicity in a process requiring mitochondrial binding of HK2. Neither HK2 nor GCK expression affects target cells' ability to activate NK cells. In contrast, mitochondrial binding of HK2 reduces effector caspase 3/7 activity both at baseline and upon NK-cell activation. Furthermore, HK2 tethering to mitochondria enhances their resistance to cytochrome c release triggered by tBID. These findings indicate that HK2 mitochondrial binding in liver cancer cells is an intrinsic resistance factor to cytolysis and an escape mechanism from immune surveillance.

Liquid biopsy: An arsenal for tumour screening and early diagnosis.

Cancer has become the second leading cause of death in the world, and more than 50% of cancer patients are diagnosed at an advanced stage. Early diagnosis of tumours is the key to improving patient quality of life and survival time and reducing the socioeconomic burden. However, there is still a lack of reliable early diagnosis methods in clinical practice. In recent years, liquid biopsy technology has developed rapidly. It has the advantages of noninvasiveness, easy access to sample sources, and reproducibility. It has become the main focus of research on the early diagnosis methods of tumours. This review summarises the research progress of existing liquid biopsy markers, such as circulating tumour DNA, circulating viral DNA, DNA methylation, circulating tumour cells, circulating RNA, exosomes, and tumour education platelets in early diagnosis of tumours, and analyses the current advantages and limitations of various markers, providing a direction for the application and transformation of liquid biopsy research in early diagnosis of clinical tumours.

Therapeutic Apheresis Using a ß2-Microglobulin Removal Column Reduces Circulating Tumor Cell Count.

An elevated serum ß2-microglobulin (ß2M) level is indicative of impaired glomerular filtration and prerenal diseases, such as malignant tumors, autoimmune disorders, and liver diseases. An elevated serum ß2M level has been shown to promote metastasis via the induction of epithelial-mesenchymal transition (EMT) in cancer cells. However, the therapeutic potential of targeting ß2M remains unclear. Here, we aimed to investigate the efficacy of Filtor, a small polymethyl methacrylate fiber-based ß2M removal column, in reducing the ß2M level and suppressing cancer cell-induced EMT and metastasis. We assessed the effects of Filtor on the changes in metastasis based on the number of circulating tumor cells (CTCs), which reflects the post-EMT cancer cell population. We performed therapeutic apheresis using Filtor on a male patient with sinonasal neuroendocrine carcinoma, a female patient with a history of colorectal cancer, and another female patient with a history of pancreatic ductal adenocarcinoma. Significantly low serum ß2M levels and CTC counts were observed immediately and 4 weeks after treatment compared with those in the pretreatment phase. Moreover, the CTC count immediately after therapeutic intervention was markedly reduced, likely because Filtor had trapped CTCs directly. These findings suggest that therapeutic apheresis with Filtor can prevent cancer metastasis and recurrence by directly removing CTCs.