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Purpose: Systemic treatments given to patients with non-small cell lung cancer (NSCLC) are often ineffective due to drug resistance. In the present study, we investigated patient-derived tumor organoids (PDTO) and matched tumor tissues from surgically treated patients with NSCLC to identify drug repurposing targets to overcome resistance toward standard-of-care platinum-based doublet chemotherapy., Experimental Design: PDTOs were established from 10 prospectively enrolled patients with non-metastatic NSCLC from resected tumors. PDTOs were compared with matched tumor tissues by histopathology/immunohistochemistry, whole exome sequencing, and transcriptome sequencing. PDTO growths and drug responses were determined by measuring 3D tumoroid volumes, cell viability, and proliferation/apoptosis. Differential gene expression analysis identified drug-repurposing targets. Validations were performed with internal/external data sets of patients with NSCLC. NSCLC cell lines were used for aldo-keto reductase 1B10 (AKR1B10) knockdown studies and xenograft models to determine the intratumoral bioavailability of epalrestat., Results: PDTOs retained histomorphology and pathological biomarker expression, mutational/transcriptomic signatures, and cellular heterogeneity of the matched tumor tissues. Five (50%) PDTOs were chemoresistant toward carboplatin/paclitaxel. Chemoresistant PDTOs and matched tumor tissues demonstrated overexpression of AKR1B10. Epalrestat, an orally available AKR1B10 inhibitor in clinical use for diabetic polyneuropathy, was repurposed to overcome chemoresistance of PDTOs. In vivo efficacy of epalrestat to overcome drug resistance corresponded to intratumoral epalrestat levels., Conclusions: PDTOs are efficient preclinical models recapitulating the tumor characteristics and are suitable for drug testing. AKR1B10 can be targeted by repurposing epalrestat to overcome chemoresistance in NSCLC. Epalrestat has the potential to advance to clinical trials in patients with drug-resistant NSCLC due to favorable toxicity, pharmacological profile, and bioavailability., (©2024 The Authors; Published by the American Association for Cancer Research.)

Extracellular vesicles (EVs) hold potential as effective carriers for drug delivery, providing a promising approach to resolving challenges in lung cancer treatment. Traditional treatments, such as with the chemotherapy drug cisplatin, encounter resistance in standard cell death pathways like apoptosis, prompting the need to explore alternative approaches. This study investigates the potential of iron oxide nanoparticles (IONP) and EVs to induce ferroptosis-a regulated cell death mechanism-in lung cancer cells. We formulated a novel EV and IONP-based system, namely 'ExoFeR', and observed that ExoFeR demonstrated efficient ferroptosis induction, evidenced by downregulation of ferroptosis markers (xCT/SLC7A11 and GPX4), increased intracellular and mitochondrial ferrous iron levels, and morphological changes in mitochondria. To enhance efficacy, tumor-targeting transferrin (TF)-conjugated ExoFeR (ExoFeR TF ) was developed. ExoFeR TF outperformed ExoFeR, exhibiting higher uptake and cell death in lung cancer cells. Mechanistically, nuclear factor erythroid 2-related factor 2 (Nrf2)-a key regulator of genes involved in glutathione biosynthesis, antioxidant responses, lipid metabolism, and iron metabolism-was found downregulated in the ferroptotic cells. Inhibition of Nrf2 intracellular translocation in ExoFeR TF -treated cells was also observed, emphasizing the role of Nrf2 in modulating ferroptosis-dependent cell death. Furthermore, ExoFeR and ExoFeR TF demonstrated the ability to sensitize chemo-resistant cancer cells, including cisplatin-resistant lung cancer patient-derived tumoroid organoids. In summary, ExoFeR TF presents a promising and multifaceted therapeutic approach for combating lung cancer by intrinsically inducing ferroptosis and sensitizing chemo-resistant cells.

Pigs are playing an increasingly vital role as translational biomedical models for studying human pathophysiology. The annotation of the pig genome was a huge step forward in translatability of pigs as a biomedical model for various human diseases. Similarities between humans and pigs in terms of anatomy, physiology, genetics, and immunology have allowed pigs to become a comprehensive preclinical model for human diseases. With a diverse range, from craniofacial and ophthalmology to reproduction, wound healing, musculoskeletal, and cancer, pigs have provided a seminal understanding of human pathophysiology. This review focuses on the current research using pigs as preclinical models for cancer research and highlights the strengths and opportunities for studying various human cancers., Competing Interests: BT is a founding member and serves as a consultant for RenOVAte Biosciences Inc. (RBI). All remaining authors declare no competing or conflict of interest., (Copyright 2024, Joshi et al.)

Cancer remains a leading cause of morbidity and mortality, and a paradigm shift is needed to fundamentally revisit drug development efforts. Pigs share close similarities to humans and may serve as an alternative model. Recently, a transgenic 'Oncopig' line has been generated to induce solid tumors with organ specificity, opening the potential of Oncopigs as a platform for developing novel therapeutic regimens., Competing Interests: Declaration of interests B.P.T. is a founding member and serves as a consultant for RenOVAte Biosciences Inc. (RBI). All remaining authors declare no competing or conflicts of interest., (Published by Elsevier Inc.)

Circulating tumor cells (CTCs) are cancer cells that slough off from the tumor and circulate in the peripheral blood and lymphatic system as micro metastases that eventually results in macro metastases. Through a simple blood draw, sensitive CTC detection from clinical samples has proven to be a useful tool for determining the prognosis of cancer. Recent technological developments now make it possible to detect CTCs reliably and repeatedly from a simple and straightforward blood test. Multicenter trials to assess the clinical value of CTCs have demonstrated the prognostic value of these cancer cells. Studies on CTCs have filled huge knowledge gap in understanding the process of metastasis since their identification in the late 19th century. However, these rare cancer cells have not been regularly used to tailor precision medicine and or identify novel druggable targets. In this review, we have attempted to summarize the milestones of CTC-based research from the time of identification to molecular characterization. Additionally, the need for a paradigm shift in dissecting these seeds of metastasis and the possible future avenues to improve CTC-based discoveries are also discussed.

Gut microbiota plays a crucial role in inflammatory bowel diseases (IBD) and can potentially prevent IBD through microbial-derived metabolites, making it a promising therapeutic avenue. Recent evidence suggests that despite an unclear underlying mechanism, red cabbage juice (RCJ) alleviates Dextran Sodium Sulfate (DSS)-induced colitis in mice. Thus, the study aims to unravel the molecular mechanism by which RCJ modulates the gut microbiota to alleviate DSS-induced colitis in mice. Using C57BL/6J mice, we evaluated RCJ's protective role in DSS-induced colitis through two cycles of 3% DSS. Mice were daily gavaged with PBS or RCJ until the endpoint, and gut microbiota composition was analyzed via shotgun metagenomics. RCJ treatment significantly improved body weight ( p ≤ 0.001), survival in mice ( p < 0.001) and reduced disease activity index (DAI) scores. Further, RCJ improved colonic barrier integrity by enhancing the expression of protective colonic mucins ( p < 0.001) and tight junction proteins ( p ≤ 0.01) in RCJ + DSS-treated mice compared to the DSS group. Shotgun metagenomic analysis revealed an enrichment of short-chain fatty acids (SCFAs)-producing bacteria ( p < 0.05), leading to increased Peroxisome Proliferator-Activated Receptor Gamma (PPAR-γ) activation ( p ≤ 0.001). This, in turn, resulted in repression of the nuclear factor κB (NFκB) signaling pathway, causing decreased production of inflammatory cytokines and chemokines. Our study demonstrates colitis remission in a DSS-induced mouse model, showcasing RCJ as a potential modulator for gut microbiota and metabolites, with promising implications for IBD prevention and treatment.

Circulating tumor cells (CTCs) that are detached from the tumor can be precursors of metastasis. The majority of studies focus on enumeration of CTCs from patient blood to predict recurrence and therapy outcomes. Very few studies have managed to expand CTCs to investigate their functional dynamics with respect to genetic changes, tumorigenic potential, and response to drug treatment. A growing amount of evidence based on successful CTC expansion has revealed novel therapeutic targets that are associated with the process of metastasis. In this review, we summarize the successes, challenges, and limitations that collectively contribute to the better understanding of metastasis using patient-derived CTCs as blood-borne seeds of metastasis. The roadblocks and future avenues to move CTC-based scientific discoveries forward are also discussed., Competing Interests: Declaration of interests All authors declare no potential conflicts of interest. All authors reviewed and approved the manuscript., (Published by Elsevier Inc.)

The interplay between western diet and gut microbiota drives the development of non-alcoholic fatty liver disease and its progression to non-alcoholic steatohepatitis. However, the specific microbial and metabolic mediators contributing to non-alcoholic steatohepatitis remain to be identified. Here, a choline-low high-fat and high-sugar diet, representing a typical western diet, named CL-HFS, successfully induces male mouse non-alcoholic steatohepatitis with some features of the human disease, such as hepatic inflammation, steatosis, and fibrosis. Metataxonomic and metabolomic studies identify Blautia producta and 2-oleoylglycerol as clinically relevant bacterial and metabolic mediators contributing to CL-HFS-induced non-alcoholic steatohepatitis. In vivo studies validate that both Blautia producta and 2-oleoylglycerol promote liver inflammation and hepatic fibrosis in normal diet- or CL-HFS-fed mice. Cellular and molecular studies reveal that the GPR119/TAK1/NF-κB/TGF-β1 signaling pathway mediates 2-oleoylglycerol-induced macrophage priming and subsequent hepatic stellate cell activation. These findings advance our understanding of non-alcoholic steatohepatitis pathogenesis and provide targets for developing microbiome/metabolite-based therapeutic strategies against non-alcoholic steatohepatitis., (© 2023. The Author(s).)

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited therapeutic options. Although immunotherapy has shown potential in TNBC patients, clinical studies have only demonstrated a modest response. Therefore, the exploration of immunotherapy in combination with chemotherapy is warranted. In this project we identified immune-related gene signatures for TNBC patients that may explain differences in patients' outcomes after anti-PD-L1+chemotherapy treatment. First, we ran the exploratory subgroup discovery algorithm on the TNBC dataset comprised of 422 patients across 24 studies. Secondly, we narrowed down the search to twelve homogenous subgroups based on tumor mutational burden (TMB, low or high), relapse status (disease-free or recurred), tumor cellularity (high, low and moderate), menopausal status (pre- or post) and tumor stage (I, II and III). For each subgroup we identified a union of the top 10% of genotypic patterns. Furthermore, we employed a multinomial regression model to predict significant genotypic patterns that would be linked to partial remission after anti-PD-L1+chemotherapy treatment. Finally, we uncovered distinct immune cell populations (T-cells, B-cells, Myeloid, NK-cells) for TNBC patients with various treatment outcomes. CD4-Tn-LEF1 and CD4-CXCL13 T-cells were linked to partial remission on anti-PD-L1+chemotherapy treatment. Our informatics pipeline may help to select better responders to chemoimmunotherapy, as well as pinpoint the underlying mechanisms of drug resistance in TNBC patients at single-cell resolution.

Rural non-small cell lung cancer (NSCLC) patients do worse, largely related to lack of access to care. In this study, the mutational characteristics and potential for targeted therapy in rural, resectable NSCLC patients using whole exome sequencing (WES) were analyzed. WES was performed on tumor-adjacent normal pairs from rural patients undergoing resection for NSCLC. Sequencing alignment, variant-calling, annotation, and tumor mutational burden (TMB) calculations were performed using standard methods. cBioportal and OncoKB were used for comparisons of mutational frequencies and actionable targets. Thirty-four NSCLC patients underwent WES after surgical resection. The gene most frequently containing somatic variants was TP53. The median number of somatic variants was 188 (Range 11-1056), and median TMB was 3.30 (0.33-18.56) nonsynonymous mutations per Mb. Tumor stage and survival were not associated with number of variants, TMB or TP53 mutational status. Significant concordance among the most common mutations when cross-referenced to cBioportal (R = 0.78, p < 0.0001) was observed. 24% of patients had variants in actionable genes based on OncoKB annotation. In summary, we demonstrate baseline mutational frequency and establish foundations for targeted adjuvant trials in rural NSCLC patients with specific differences. Future studies must ensure to include rural patients to improve NSCLC patient outcomes., Competing Interests: Declaration of Competing Interest A.A.C. has patent filings related to cancer biomarkers, and has licensed technology to Droplet Biosciences, Tempus Labs and to Biocognitive Labs. A.A.C. has served as a consultant/advisor to Roche, Tempus, Geneoscopy, NuProbe, Daiichi Sankyo, AstraZeneca, AlphaSights, and Guidepoint. A.A.C. has received honoraria from Roche, Foundation Medicine, and Dava Oncology. A.A.C. has stock options in Geneoscopy, research support from Roche and Tempus Labs, and ownership interests in Droplet Biosciences and LiquidCell Dx. J.T.K. has patent filings related to cancer biomarkers. None of the other authors have any conflict of interest in regard to the work presented in this manuscript., (Copyright © 2022. Published by Elsevier Inc.)

Cytotoxic T lymphocyte (CTL) infiltration is associated with survival, recurrence, and therapeutic response in colorectal cancer (CRC). Immune checkpoint inhibitor (ICI) therapy, which requires CTLs for response, does not work for most CRC patients. Therefore, it is critical to improve our understanding of immune resistance in this disease. We utilized 2391 CRC patients and 7 omics datasets, integrating clinical and genomic data to determine how DNA methylation may impact survival and CTL function in CRC. Using comprehensive molecular subtype (CMS) 1 patients as reference, we found TBX21 to be the only gene with altered expression and methylation that was associated with CTL infiltration. We found that CMS1 patients with high TBX21 expression and low methylation had a significant survival advantage. To confirm the role of Tbx21 in CTL function, we utilized scRNAseq data, demonstrating the association of TBX21 with markers of enhanced CTL function. Further analysis using pathway enrichment found that the genes TBX21, MX1, and SP140 had altered expression and methylation, suggesting that the TP53/P53 pathway may modify TBX21 methylation to upregulate TBX21 expression. Together, this suggests that targeting epigenetic modification more specifically for therapy and patient stratification may provide improved outcomes in CRC.

The epigenetic regulation of immune response involves reversible and heritable changes that do not alter the DNA sequence. Though there have been extensive studies accomplished relating to epigenetic changes in cancer cells, recent focus has been shifted on epigenetic-mediated changes in the immune cells including T cells, Macrophages, Natural Killer cells and anti-tumor immune responses. This review compiles the most relevant and recent literature related to the role of epigenetic mechanisms including DNA methylation and histone modifications in immune cells of wide range of cancers. We also include recent research with respect to role of the most relevant transcription factors that epigenetically control the anti-tumor immune response. Finally, a statement of future direction that promises to look forward for strategies to improve immunotherapy in cancer., (Copyright © 2021. Published by Elsevier Ltd.)

Low-dose computed tomography (LDCT) Non-Small Cell Lung (NSCLC) screening is associated with high false-positive rates, leading to unnecessary expensive and invasive follow ups. There is a need for minimally invasive approaches to improve the accuracy of NSCLC diagnosis. In addition, NSCLC patients harboring sensitizing mutations in epidermal growth factor receptor EGFR (T790M, L578R) are treated with Osimertinib, a potent tyrosine kinase inhibitor (TKI). However, nearly all patients develop TKI resistance. The underlying mechanisms are not fully understood. Plasma extracellular vesicle (EV) and circulating microRNA (miRNA) have been proposed as biomarkers for cancer screening and to inform treatment decisions. However, the identification of highly sensitive and broadly predictive core miRNA signatures remains a challenge. Also, how these systemic and diverse miRNAs impact cancer drug response is not well understood. Using an integrative approach, we examined plasma EV and circulating miRNA isolated from NSCLC patients versus screening controls with a similar risk profile. We found that combining EV (Hsa-miR-184, Let-7b-5p) and circulating (Hsa-miR-22-3p) miRNAs abundance robustly discriminates between NSCLC patients and high-risk cancer-free controls. Further, we found that Hsa-miR-22-3p, Hsa-miR-184, and Let-7b-5p functionally converge on WNT/βcatenin and mTOR/AKT signaling axes, known cancer therapy resistance signals. Targeting Hsa-miR-22-3p and Hsa-miR-184 desensitized EGFR-mutated (T790M, L578R) NSCLC cells to Osimertinib. These findings suggest that the expression levels of circulating hsa-miR-22-3p combined with EV hsa-miR-184 and Let-7b-5p levels potentially define a core biomarker signature for improving the accuracy of NSCLC diagnosis. Importantly, these biomarkers have the potential to enable prospective identification of patients who are at risk of responding poorly to Osimertinib alone but likely to benefit from Osimertinib/AKT blockade combination treatments., (© 2022. The Author(s).)

Combination therapy represents an effective therapeutic approach to overcome hepatocellular cancer (HCC) resistance to immune checkpoint blockade (ICB). Based upon previous work demonstrating that nanoliposome C6-ceramide (LipC6) not only induces HCC apoptosis but also prevents HCC-induced immune tolerance, we now investigate the potential of LipC6 in combination with ICB in HCC treatment. We generated orthotopic HCC-bearing mice, which have typical features in common with human patients, and then treated them with LipC6 in combination with the antibodies (Abs) for programmed cell death protein 1 (PD-1) or cytotoxic T-lymphocyte antigen 4 (CTLA4). The tumor growth was monitored by magnetic resonance imaging (MRI) and the intrahepatic immune profiles were checked by flow cytometry in response to the treatments. Realtime PCR (qPCR) was used to detect the expression of target genes. The results show that LipC6 in combination with anti-CTLA4 Ab, but not anti-PD-1 Ab, significantly slowed tumor growth, enhanced tumor-infiltrating CD8 + T cells, and suppressed tumor-resident CD4 + CD25 + FoxP3 + Tregs. Further molecular investigation indicates that the combinational treatment suppressed transcriptional factor Krüppel-like Factor 2 (KLF2), forkhead box protein P3 (FoxP3), and CTLA4. Our studies suggest that LipC6 in combination with anti-CTLA4 Ab represents a novel therapeutic approach with significant potential in activating anti-HCC immune response and suppressing HCC growth., (© 2022 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Purpose: Low-dose computed tomography (LDCT) screening of high-risk patients decreases lung cancer-related mortality. However, high false-positive rates associated with LDCT result in unnecessary interventions. To distinguish non-small-cell lung cancer (NSCLC) from benign nodules, in the present study, we integrated cellular liquid biomarkers in patients with suspicious lung nodules (lung cancer screening reporting and data system [Lung-RADS] 4)., Methods: Prospectively, 7.5 mL of blood was collected from 221 individuals (training set: 90 nonscreened NSCLC patients, 74 high-risk screening patients with no/benign nodules [Lung-RADS 1-3], and 20 never smokers; validation set: 37 patients with suspicious nodules [Lung-RADS 4]). Circulating tumor cells (CTCs), CTC clusters, and tumor-macrophage fusion (TMF) cells were identified by blinded analyses. Screening patients underwent a median of two LDCTs (range, 1-4) with a median surveillance time of 30 (range, 11-50) months., Results: In the validation set of 37 Lung-RADS 4 patients, all circulating cellular biomarker counts ( P < .005; Wilcoxon test) and positivity rates were significantly higher in 23 biopsy-proven NSCLC patients (CTCs: 23 of 23 [100%], CTC clusters: 6 of 23 [26.1%], and TMF cells: 15 of 23 [65.2%]) than in 14 patients with biopsy-proven benign nodules (6 of 14 [42.9%], 0 of 14 [0%], and 2 of 14 [14.3%]). On the basis of cutoff values from the training set, logistic regression with receiver operating characteristic and area under the curve analyses demonstrated that CTCs (sensitivity: 0.870, specificity: 1.0, and area under the curve: 0.989) and TMF cells (0.652; 0.880; 0.790) complement LDCT in diagnosing NSCLC in Lung-RADS 4 patients., Conclusion: Cellular liquid biomarkers have a potential to complement LDCT interpretation of suspicious Lung-RADS 4 nodules to distinguish NSCLC from benign lung nodules. A future prospective, large-scale, multicenter clinical trial should validate the role of cellular liquid biomarkers in improving diagnostic accuracy in high-risk patients with Lung-RADS 4 nodules., Competing Interests: Kevin F. Staveley-O'CarrollHonoraria: AstraZeneca Klaus PantelHonoraria: Agena Bioscience, Novartis, Roche, Medac, Impulze, Ipsen, Sanofi, Merck KGaA, MSD, Beiersdorf, Galderma, Hummingbird, Illumina, Hello Healthcare, Menarini Silicon Biosystems, Abcam, Atheneum, CureVac, DeciBio, Inflection Biosciences, Molecular Health, TacticsConsulting or Advisory Role: Sanofi, Agena Bioscience, Hummingbird Diagnostics, Menarini Silicon BiosystemsResearch Funding: Janssen Diagnostics, Cancer-ID (Inst)Patents, Royalties, Other Intellectual Property: Application No. WO2016 128125A1, application No. 17157020.3—1405, application No. 10004180.5, application No. 07825055.2, application No. 95108760.7Travel, Accommodations, Expenses: Agena Bioscience Guangfu LiPatents, Royalties, Other Intellectual Property: Ceramide nanoliposomes as a method and device for immunotherapy (Inst) Aadel A. ChaudhuriLeadership: Droplet BiosciencesStock and Other Ownership Interests: Geneoscopy, Droplet BiosciencesHonoraria: RocheConsulting or Advisory Role: Geneoscopy, Roche, Tempus, AstraZeneca/Daiichi SankyoPatents, Royalties, Other Intellectual Property: US Patent No. US8685727B2Travel, Accommodations, Expenses: Roche, Foundation MedicineOther Relationship: Roche Jussuf T. KaifiPatents, Royalties, Other Intellectual Property: Cancer Biomarker detectionNo other potential conflicts of interest were reported.

Non-small-cell lung cancer (NSCLC) accounts for most cancer-related deaths worldwide. Liquid biopsy by a blood draw to detect circulating tumor cells (CTCs) is a tool for molecular profiling of cancer using single-cell and next-generation sequencing (NGS) technologies. The aim of the study was to identify somatic variants in single CTCs isolated from NSCLC patients by targeted NGS. Thirty-one subjects (20 NSCLC patients, 11 smokers without cancer) were enrolled for blood draws (7.5 mL). CTCs were identified by immunofluorescence, individually retrieved, and DNA-extracted. Targeted NGS was performed to detect somatic variants (single-nucleotide variants (SNVs) and insertions/deletions (Indels)) across 65 oncogenes and tumor suppressor genes. Cancer-associated variants were classified using OncoKB database. NSCLC patients had significantly higher CTC counts than control smokers ( p = 0.0132; Mann-Whitney test). Analyzing 23 CTCs and 13 white blood cells across seven patients revealed a total of 644 somatic variants that occurred in all CTCs within the same subject, ranging from 1 to 137 per patient. The highest number of variants detected in ≥1 CTC within a patient was 441. A total of 18/65 (27.7%) genes were highly mutated. Mutations with oncogenic impact were identified in functional domains of seven oncogenes/tumor suppressor genes ( NF1, PTCH1, TP53, SMARCB1, SMAD4, KRAS, and ERBB2 ). Single CTC-targeted NGS detects heterogeneous and shared mutational signatures within and between NSCLC patients. CTC single-cell genomics have potential for integration in NSCLC precision oncology.

Pancreatic cancer (PaC) is resistant to immune checkpoint therapy, but the underlying mechanisms are largely unknown. In this study, we have established four orthotopic PaC murine models with different PaC cell lines by intra-pancreatic inoculation. Therapeutic examinations demonstrate that only tumors induced with Panc02-H7 cells respond to αPD-1 antibody treatment, leading to significantly reduced tumor growth and increased survival in the recipient mice. Transcriptomic profiling at a single-cell resolution characterizes the molecular activity of different cells within tumors. Comparative analysis and validated experiments demonstrate that αPD-1-sensitive and -resistant tumors differently shape the immune landscape in the tumor microenvironment (TME) and markedly altering effector CD8 + T cells and tumor-associated macrophages (TAMs) in their number, frequency, and gene profile. More exhausted effector CD8 + T cells and increased M2-like TAMs with a reduced capacity of antigen presentation are detected in resistant Panc02-formed tumors versus responsive Panc02-H7-formed tumors. Together, our data highlight the correlation of tumor-induced imbalance of macrophages with the fate of tumor-resident effector CD8 + T cells and PaC response to αPD-1 immunotherapy. TAMs as a critical regulator of tumor immunity and immunotherapy contribute to PaC resistance to immune checkpoint blockade., (Copyright © 2021. Published by Elsevier Inc.)

Microfluidics have been applied to filtration of rare tumor cells from the blood as liquid biopsies. Processing is highly limited by low flow rates and device clogging due to a single function of fluidic paths. A novel method using multifunctional hybrid functional microposts was developed. A swift by-passing route for non-tumor cells was integrated to prevent very common clogging problems. Performance was characterized using microbeads (10 µm) and human cancer cells that were spiked in human blood. Design-I showed a capture efficiency of 96% for microbeads and 87% for cancer cells at 1 ml/min flow rate. An improved Design-II presented a higher capture efficiency of 100% for microbeads and 96% for cancer cells. Our method of utilizing various microfluidic functions of separation, bypass and capture has successfully guaranteed highly efficient separation of rare cells from biological fluids., (© 2021. The Author(s).)

Background: Lung cancer metastases to the breast are less common and consequently have received much less attention in clinical practice. The purpose of this study was to provide a better understanding of clinical, ultrasonographic, and immunohistochemical features of breast metastases from primary lung cancer., Methods: This retrospective case series included patients with breast metastases from primary lung cancer between January 2012 and December 2020. Clinical features, ultrasonographic characteristics, and immunohistochemical findings were evaluated in this analysis., Results: In all, 7 cases (mean ± standard deviation age: 57.4±8.3 years; range, 49-70 years) were evaluated. The maximum size of breast lesions in 6 cases ranged from 1.2 to 4.5 cm, while 1 case showed a diffused pattern. Ultrasound features of breast metastases from lung cancer were irregular (5/7, 71.4%), indistinct (6/7, 85.7%), hypoechoic (7/7, 100.0%), and parallel (6/7, 85.7%) masses without calcification. Immunohistochemical staining test was positive for thyroid transcription factor 1 (TTF-1) in all patients (7/7, 100.0%), 3 cases (3/5, 60.0%) were negative for p63, 5 cases (5/5, 100.0%) were positive for cytokeratin 7 (CK7), 4 cases (4/5, 80.0%) were positive for napsin A., Conclusions: The ultrasonographic features of lung metastases to the breast are clinically important to understand. A known history of the primary lung cancer is of great importance when evaluating patients with a breast nodule. The presence of an ipsilateral lung cancer, breast nodule and axillary lymphadenopathy should be considered with pathological and immunohistochemical data to differentiate breast metastases from a primary breast malignancy in this setting., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://dx.doi.org/10.21037/tlcr-21-542). The authors have no conflicts of interest to declare., (2021 Translational Lung Cancer Research. All rights reserved.)

Enabling precision medicine requires developing robust patient stratification methods as well as drugs tailored to homogeneous subgroups of patients from a heterogeneous population. Developing de novo drugs is expensive and time consuming with an ultimately low FDA approval rate. These limitations make developing new drugs for a small portion of a disease population unfeasible. Therefore, drug repositioning is an essential alternative for developing new drugs for a disease subpopulation. This shows the importance of developing data-driven approaches that find druggable homogeneous subgroups within the disease population and reposition the drugs for these subgroups. In this study, we developed an explainable AI approach for patient stratification and drug repositioning. Contrast pattern mining and network analysis were used to discover homogeneous subgroups within a disease population. For each subgroup, a biomedical network analysis was done to find the drugs that are most relevant to a given subgroup of patients. The set of candidate drugs for each subgroup was ranked using an aggregated drug score assigned to each drug. The proposed method represents a human-in-the-loop framework, where medical experts use the data-driven results to generate hypotheses and obtain insights into potential therapeutic candidates for patients who belong to a subgroup. Colorectal cancer (CRC) was used as a case study. Patients' phenotypic and genotypic data was utilized with a heterogeneous knowledge base because it gives a multi-view perspective for finding new indications for drugs outside of their original use. Our analysis of the top candidate drugs for the subgroups identified by medical experts showed that most of these drugs are cancer-related, and most of them have the potential to be a CRC regimen based on studies in the literature., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Colorectal cancer (CRC) remains one of the deadliest malignancies worldwide despite recent progress in treatment strategies. Though immune checkpoint inhibition has proven effective for a number of other tumors, it offers benefits in only a small group of CRC patients with high microsatellite instability. In general, heterogenous cell groups in the tumor microenvironment are considered as the major barrier for unveiling the causes of low immune response. Therefore, deconvolution of cellular components in highly heterogeneous microenvironments is crucial for understanding the immune contexture of cancer. In this review, we assimilate current knowledge and recent studies examining anti-tumor immunity in CRC. We also discuss the utilization of novel immune contexture assessment methods that have not been used in CRC research to date.

Pancreatic cancer (PC) is one of the most lethal human malignancies without effective treatment. In an effort to discover key genes and molecular pathways underlying PC growth, we have identified LIM domain only 7 (LMO7) as an under-investigated molecule, which highly expresses in primary and metastatic human and mouse PC with the potential of impacting PC tumorigenesis and metastasis. Using genetic methods with siRNA, shRNA, and CRISPR-Cas9, we have successfully generated stable mouse PC cells with LMO7 knockdown or knockout. Using these cells with loss of LMO7 function, we have demonstrated that intrinsic LMO7 defect significantly suppresses PC cell proliferation, anchorage-free colony formation, and mobility in vitro and slows orthotopic PC tumor growth and metastasis in vivo . Mechanistic studies demonstrated that loss of LMO7 function causes PC cell-cycle arrest and apoptosis. These data indicate that LMO7 functions as an independent and unrecognized druggable factor significantly impacting PC growth and metastasis, which could be harnessed for developing a new targeted therapy for PC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Liu, Yuan, Zhou, Wang, Qi, Bernal, Avella, Kaifi, Kimchi, Timothy, Cheng, Miao, Jiang and Li.)

Background: Minimally invasive radiofrequency ablation (RFA) is used as a first-line treatment option for hepatocellular cancer (HCC) with the weaknesses of incomplete ablation, tumor recurrence, and inferior outcomes. To overcome this limitation, we proposed to develop sunitinib-RFA integrated therapy with a potential of activating anti-HCC immune response., Methods: Using our unique murine model, we developed a novel RFA platform with a modified human cardiac RF generator. Therapeutic efficacy of sunitinib-RFA combined treatment in HCC was tested in this platform. Tumor progression was monitored by MRI; tumor necrosis and apoptosis were detected by H&E and terminal deoxynucleotidyl transferase dUTP nick end labeling; immune reaction was defined by flow cytometry; and signaling molecules were examined with real-time PCR (qPCR), western blot, and immunohistochemical staining., Results: A significantly reduced tumor growth and extended lift span were observed in the mice receiving combined treatment with RFA and sunitinib. This combined treatment significantly increased the frequency of CD8 + T cell, memory CD8 + T cell, and dendritic cells (DCs); decreased the frequency of regulatory T cells; and activated tumor-specific antigen (TSA) immune response in tumor microenvironment. We found that RFA caused PD-1 upregulation in tumor-infiltrated T cells by boosting hepatocyte growth factor (HGF) expression, which was suppressed by sunitinib treatment. We have also demonstrated that sunitinib suppressed VEGF's effect in enhancing PD-L1 expression in DCs and attenuated heat-sink effect. The results indicate that RFA induced tumor destruction and release of in situ TSAs which can activate a tumoricidal immune response in sunitinib-treated mice, significantly improving anti-HCC therapeutic efficacy., Conclusions: Sunitinib enables RFA-released in situ TSA to ignite an effective anti-tumor immune response by suppressing HGF and VEGF signaling pathways. Sunitinib-RFA as a synergistic therapeutic approach significantly suppresses HCC growth., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Introduction: Various subtypes of circulating cancer-associated cells in the blood are described. A unique circulating, large, and polymorphonuclear cell with a dual epithelial and myeloid phenotype has been suggested as a tumor-macrophage fusion cell (TMF). The goal of the study was to identify the impact of distinct TMFs on survival among patients with NSCLC., Methods: In this prospective trial, 7.5 mL of whole blood sample was collected. After microfilter enrichment, immunofluorescent staining was performed, identifying TMFs as greater than or equal to 30 μm in size and dual epithelial (cytokeratin 8, 18, or 19-, or epithelial cell adhesion molecule-positive) and myeloid- or macrophage-positive (CD14- or CD45-positive) cells with at least one 4',6-diamidino-2-phenylindole+ nucleus., Results: Circulating TMFs were identified in 88 of 115 patients (76.5%) with NSCLC (mean 3.052 [SEM ± 0.306]; median 2 [range 0-17]) but were rare in long-term smokers without cancer (6 of 87 [6.9%]; 0.081 [±0.034]; 0 [0-2]), and absent in 20 healthy controls. Comparing the presence of TMFs in patients with NSCLC versus smokers without cancer, specificity was 93.1% (95% confidence interval: 85.6-97.4%) and sensitivity 76.5% (95% confidence interval: 67.7%-83.9%). TMF counts correlated with American Joint Committee on Cancer tumor stages. More importantly, more than one TMF and giant TMFs sizes greater than or equal to 50 μm were associated with statistically significantly shorter overall and cancer-specific disease-free (p < 0.05) survival after curative resection for stage I to IIIA. Giant TMFs greater than or equal to 50 μm size were an independent survival predictor by multivariate analysis., Conclusions: Circulating, in particular, giant TMFs are associated with aggressive clinical behavior in surgically treated patients with NSCLC. The biological role of unique TMFs will need to be further investigated, as these may have a potential impact on immune responses toward cancer., (Published by Elsevier Inc.)

Objectives: Over 25% of the high-risk population screened for lung cancer have an abnormal computed tomography (CT) scan. Conventionally, these lesions have been biopsied with CT guidance with a high diagnostic yield. Electromagnetic navigational bronchoscopy (ENB) with transbronchial biopsy has emerged as a technology that improves the diagnostic sensitivity of conventional bronchoscopic biopsy. It has been used to biopsy lung lesions, due to the low risk of pneumothorax. It is, however, a new technology that is expensive and its role in the diagnosis of the solitary pulmonary nodule (SPN) is yet to be determined. The purpose of this study was to evaluate the diagnostic yield of CT-guided biopsy (CTB) following non-diagnostic ENB biopsy and identify characteristics of the lesion that predicts a low diagnostic yield with ENB, to ensure appropriate use of ENB in the evaluation of SPN., Materials and Methods: One hundred and thirty-five lung lesions were biopsied with ENB from January 2017 to August 2019. Biopsies were considered diagnostic if pathology confirmed malignancy or inflammation in the appropriate clinical and imaging setting. We evaluated lesions for several characteristics including size, lobe, and central/peripheral distribution. The diagnostic yield of CTB in patients who failed ENB biopsies was also evaluated. Logistic regression was used to identify factors likely to predict a non-diagnostic ENB biopsy., Result: Overall, ENB biopsies were performed in 135 patients with solitary lung lesions. ENB biopsies were diagnostic in 52% (70/135) of the patients. In 23 patients with solitary lung lesions, CTBs were performed following a non-diagnostic ENB biopsy. The CTBs were diagnostic in 87% of the patients (20/23). ENB biopsies of lesions <21.5 mm were non-diagnostic in 71% of cases (42/59); 14 of these patients with non-diagnostic ENB biopsies had CTBs, and 86% of them were diagnostic (12/14). ENB biopsies of lesions in the lower lobes were non- diagnostic in 59% of cases (35/59); 12 of these patients with non-diagnostic ENB biopsies had CTBs, and 83% were diagnostic (10/12). ENB biopsies of lesions in the outer 2/3 were non-diagnostic in 57% of cases (50/87); 21 of these patients with non-diagnostic ENB biopsies had CTBs, and 86% were diagnostic (18/21)., Conclusion: CTBs have a high diagnostic yield even following non-diagnostic ENB biopsies. Lesions <21.5 mm, in the outer 2/3 of the lung, and in the lower lung have the lowest likelihood of a diagnostic yield with ENB biopsies. Although CTBs have a slightly higher pneumothorax rate, these lesions would be more successfully diagnosed with CTB as opposed to ENB biopsy, in the process expediting the diagnosis and saving valuable medical resources., Competing Interests: There are no conflicts of interest., (© 2020 Published by Scientific Scholar on behalf of Journal of Clinical Imaging Science.)

Background: In non-small cell lung cancer (NSCLC), 18 F-fluoro-2-deoxy-D-glucose ( 18 F-FDG) uptake determined by PET and presence of circulating tumor cells (CTCs) in the peripheral blood independently predict outcomes. For 18 F-FDG PET/CT staging interpretation, standardized uptake values (SUV max/avg ) are routinely used in clinical reporting. The goal was to investigate whether 18 F-FDG uptake measured by SUV max/avg , but also measures of metabolic tumor volume (MTV) and total lesion glycolysis (TLG) (MTV × SUV avg ), are associated with CTCs., Methods: Prospectively, 7.5 mL blood was drawn from NSCLC patients at the time of staging 18F-FDG PET/CT and from healthy control subjects. CTCs were identified by immunofluorescent staining (CK8/18/19 pos /EpCAM pos /CD45 neg /DAPI pos nucleus). 18 F-FDG PET/CTs were analyzed for SUV max , SUV avg , MTV, and TLG., Results: In 16 NSCLC patients with stage I-IIIA, MTV and TLG, in contrast to SUV max and SUV avg , were positively associated with CTCs (linear regression analysis). No CTCs were detectable in 20 healthy control subjects., Conclusions: This pilot study demonstrates that 18 F-FDG PET/CT TLG correlates with CTCs in NSCLC patients without distant metastases. TLG might be a more appropriate marker for hematogenous micrometastatic potential than SUVs., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/tlcr.2020.04.10). The authors have no conflicts of interest to declare., (2020 Translational Lung Cancer Research. All rights reserved.)

Although molecular mechanisms driving tumor progression have been extensively studied, the biological nature of the various populations of circulating tumor cells (CTCs) within the blood is still not well understood. Tumor cell fusion with immune cells is a longstanding hypothesis that has caught more attention in recent times. Specifically, fusion of tumor cells with macrophages might lead to the development of metastasis by acquiring features such as genetic and epigenetic heterogeneity, chemotherapeutic resistance, and immune tolerance. In addition to the traditional FDA-approved definition of a CTC (CD45-, EpCAM+, cytokeratins 8+, 18+ or 19+, with a DAPI+ nucleus), an additional circulating cell population has been identified as being potential fusions cells, characterized by distinct, large, polymorphonuclear cancer-associated cells with a dual epithelial and macrophage/myeloid phenotype. Artificial fusion of tumor cells with macrophages leads to migratory, invasive, and metastatic phenotypes. Further studies might investigate whether these have a potential impact on the immune response towards the cancer. In this review, the background, evidence, and potential relevance of tumor cell fusions with macrophages is discussed, along with the potential role of intercellular connections in their formation. Such fusion cells could be a key component in cancer metastasis, and therefore, evolve as a diagnostic and therapeutic target in cancer precision medicine.

Exosomes are extracellular vesicles which are released from healthy and tumor cells into blood circulation. Unique biomolecular cargos such as RNA and protein are loaded in these vesicles. These molecules may have biological functions such as signaling, cell communications and have the potential to be analyzed as biomarkers. In this initial study, we describe the analysis of exosomes in the serum of healthy subjects, intraductal papillary mucosal neoplasms and pancreatic ductal adenocarcinoma including the characterization of their RNA cargos by next generation sequencing (EXO-NGS). Results indicate the presence of a wide variety of RNAs including mRNA, miRNA, lincRNA, tRNA and piRNA in these vesicles. Based on the differential mRNA expression observed upon EXO-NGS analysis, we independently evaluated two protein coding genes, matrix metalloproteinase-8 (MMP-8) and transcription factor T-Box 3 (TBX3) by qRT-PCR for selective expression in the serum samples. Results indicate a variable expression pattern of these genes across serum samples between different study groups. Further, qRT-PCR analysis with the same serum exosomes processed for EXO-NGS, we observed two long non-coding RNAs, malat-1 and CRNDE to be variably expressed. Overall, our observations emphasize the potential value of different exosome components in distinguishing between healthy, premalignant and malignant conditions related to the pancreas.

PURPOSE Post-thoracotomy pain syndrome is a common condition affecting up to 50% of post-thoracotomy patients. However, percutaneous computed tomography (CT)-guided intercostal nerve cryoablation may provide symptomatic benefit in chronic and/or refractory cases. METHODS A retrospective review of our institution's comprehensive case log from October 2017 to September 2018 for patients who underwent cryoablation was analyzed. Thirteen patients with post-thoracotomy pain syndrome, refractory to medical management, were treated with CT-guided intercostal nerve cryoablation. Most patients had treatment of the intercostal nerve at the level of their thoracotomy scar, two levels above and below. The safety and technical success of this technique and the clinical outcomes of the study population were then retrospectively reviewed. RESULTS Of the patients, 69% experienced significant improvement in their pain symptoms with a median pain improvement score of 3 points (range, -1 to 8 points) over a median follow-up of 11 months (range, 2-18.6 months). Complications included pneumothorax in 8% and pseudohernia in 23% of patients. CONCLUSION CT-guided intercostal nerve cryoablation may be an effective technique in the treatment of post-thoracotomy pain syndrome and requires further study.

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded elucidation of the underlying mechanisms and development of innovative immunotherapeutic strategies. To develop an ideal animal model recapitulating human HCC, immunocompetent male C57BL/6J mice first receive a carbon tetrachloride (CCl4) injection to induce liver fibrosis, then receive histologically-normal oncogenic hepatocytes from young male SV40 T antigen (TAg)-transgenic mice (MTD2) by intra-splenic (ISPL) inoculation. Androgen generated in recipient male mice at puberty initiates TAg expression under control of a liver-specific promoter. As a result, the transferred hepatocytes become cancer cells and form tumor masses in the setting of liver fibrosis/cirrhosis. This novel model mimics human HCC initiation and progression in the context of liver fibrosis/cirrhosis and reflects the most typical features of human HCC including immune dysfunction.

Objectives: Circulating tumor cell (CTC) clusters (≥2 CTCs in aggregate) detected in the peripheral blood have predictive value in solid cancers, including non-small cell lung cancer (NSCLC). The goal of the study was to investigate the presence of CTC clusters in NSCLC patients and in high-risk screening subjects having no or benign nodules in a screening low-dose CT (LDCT)., Materials and Methods: In a prospective pilot trial, 7.5 ml peripheral blood was collected from treatment-naïve NSCLC patients, LDCT screening subjects (55-80 years, ≥30 pack-year smoking history) with no (Lung-RADS 1) or benign lung nodules (Lung-RADS 2), and healthy never-smoking controls. CTCs were enriched by size, also allowing CTC cluster isolation. For CTC identification and enumeration, immunofluorescence staining was performed for cytokeratins (CK) 8/18 and/or 19, EpCAM, CD45, and nuclei were stained with DAPI. Clinicopathological data were collected, and LDCT interpreted by the American College of Radiology Lung-RADS criteria., Results: CTC clusters were detected in 12/29 (41.4%) of all NSCLC patients, but not found in 31 high-risk screening subjects with Lung-RADS 1 or Lung-RADS 2 (P < 0.05). Since non-clustered, single CTCs were detectable in both groups of NSCLC patients (100%) and in 18/31 (58.1%) of high-risk screening subjects. No CTCs were detected in 20 healthy control subjects., Conclusion: This pilot study suggests that CTC clusters are a useful and specific liquid biomarker to further explore for screening by LDCT and risk stratification of NSCLC patients. Future prospective studies with higher subject numbers will need to be performed., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Liver-intestine cadherin (CDH17) has been known to function as a tumor stimulator and diagnostic marker for almost two decades. However, its function in highly malignant pancreatic cancer (PC) has yet to be elucidated. Using different strategies including siRNA, shRNA, and CRISPR technology, we successfully induced knockdown and knockout of CDH17 in Panc02-H7 cells and established the corresponding stable cell lines. With these cells, we demonstrated that loss of CDH17 function not only suppressed Panc02-H7 cell growth in vitro but also significantly slowed orthotopic tumor growth in vivo, resulting in the significant life extension. In vitro studies demonstrated that impairing CDH17 inhibited cell proliferation, colony formation, and motility by mechanistically modulating pro- and anti-apoptosis events in PC cells, as CDH17 suppression obviously increased expression of Bad, cytochrome C, cleaved caspase 3, and cleaved PARP, and reduced expression of Bcl-2, Survivin, and pAkt. In vivo studies showed CDH17 knockout resulted in apoptotic PC tumor death through activating caspase-3 activity. Taken together, CDH17 functions as an oncogenic molecule critical to PC growth by regulating tumor apoptosis signaling pathways and CDH17 could be targeted to develop an anti-PC therapeutic approach., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In addition to the FDA-approved definition of a circulating tumor cell (CTC), various CTC phenotypes have been discovered. Epithelial-mesenchymal transition (EMT) of cancer cells is directly linked to PD-L1 upregulation. The goal of the study was to investigate PD-L1 expression and EMT in CTCs of non-small cell lung cancer (NSCLC) patients, and perform an outcome analysis. Prospectively, 7.5 mL peripheral blood was collected from 30 NSCLC patients that underwent surgery and 15 healthy controls. CTCs were enriched by size-based microfilter and immunofluorescence stainings performed (cytokeratin (CK) 8/18/19, EpCAM, CD45, PD-L1, EMT markers vimentin, and N-Cadherin, DAPI). Patient-matched NSCLC tissues were also stained. CTC staining intensity was quantified with a software and correlated with patient-matched NSCLC tissues and survival. PD-L1 and EMT markers were expressed at significantly higher proportions in CTCs than patient-matched NSCLC tissues ( p < 0.05); ≥3 PD-L1 pos /EMT pos CTCs were associated with significantly poorer survival after curative surgery ( p < 0.05). No CTCs were detected in 15 healthy controls. This study shows that PD-L1 expression and EMT of CTCs is a negative survival predictor for NSCLC patients. The therapeutic role of the molecular linkage of PD-L1 and EMT will need to be further investigated, as linked pathways could be targeted to improve NSCLC outcome.