This review examines the clinical application of label-free microscopy and spectroscopy, which are based on optical signals emitted by tissue components. Over the past three decades, a variety of techniques have been investigated with the aim of developing an in situ histopathology method that can rapidly and accurately identify tumor margins during surgical procedures. These techniques can be divided into two groups. One group encompasses techniques exploiting linear optical signals, and includes infrared and Raman microspectroscopy, and autofluorescence microscopy. The second group includes techniques based on nonlinear optical signals, including harmonic generation, coherent Raman scattering, and multiphoton autofluorescence microscopy. Some of these methods provide comparable information, while others are complementary. However, all of them have distinct advantages and disadvantages due to their inherent nature. The first part of the review provides an explanation of the underlying physics of the excitation mechanisms and a description of the instrumentation. It also covers endomicroscopy and data analysis, which are important for understanding the current limitations in implementing label-free techniques in clinical settings. The second part of the review describes the application of label-free microscopy imaging to improve oncological surgery with focus on brain tumors and selected gastrointestinal cancers, and provides a critical assessment of the current state of translation of these methods into clinical practice. Finally, the potential of confocal laser endomicroscopy for the acquisition of autofluorescence is discussed in the context of immediate clinical applications.

We report the synthesis and characterization of a new 4-methoxyphenyl-2,2'-bipyridine-based ligand, such as 12, bearing dipicolylaminomethyl core as a receptor unit, as a probe for the fluorescence "turn-on" detection of Zn2+. Thus, in the presence of Zn2+ the probe 12 exhibited a fluorescence enhancement with a Stokes shift of ~ 180 nm and photoluminescence quantum yields value of ~ 1.0. In addition, 12 exhibited higher binding constant for Zn2+ (~ 2 × 105 M-1) with the LOD reaching the nanomolar level (~ 0.1 × 10-9 M) compare to the previously reported probe 1. The stoichiometry and structure of the [Zn(12)]2+ and [Zn(1)]2+ complexes were supported by XRD analysis, DFT calculations and 1H NMR experiments. It was postulated that, as a result of binding of Zn2+, the sample exhibited a bright "on" state via the PET-ICT processes. Molecular docking studies and confocal fluorescence microscopy experiments demonstrated that the probe 12 could be used for the fluorescence detection of Zn2+ not only in artificially enriched with zinc salts live cells, but also in fixed tissues with cations are in a bound state. The high binding constant of compound 12 to Zn2+ cation allows it to be used for the accurate localization of pancreatic beta cells (islets of Langerhans).

The existence of cancer stem cells (CSCs) in various tumors has become increasingly clear in addition to their prominent role in therapy resistance, metastasis, and recurrence. For early diagnosis, disease progression monitoring, and targeting, there is a high demand for clinical-grade methods for quantitative measurement of CSCs from patient samples. Despite years of active research, standard measurement of CSCs has not yet reached clinical settings, especially in the case of solid tumors. This is because detecting this plastic heterogeneous population of cells is not straightforward. This review summarizes various techniques, highlighting their benefits and limitations in detecting CSCs from patient samples. In addition, methods designed to detect CSCs based on secreted and niche-associated signaling factors are reviewed. Spatial and single-cell methods for analyzing patient tumor tissues and noninvasive techniques such as liquid biopsy and in vivo imaging are discussed. Additionally, methods recently established in laboratories, preclinical studies, and clinical assays are covered. Finally, we discuss the characteristics of an ideal method as we look toward the future.

Pancreatic intraepithelial neoplasia (PanIN) is the most common precursor lesion of pancreatic ductal adenocarcinoma (PDAC), which has poor prognosis with a short median overall survival of 6-12 months and a low 5-year survival rate of approximately 3%. It is crucial to remove PanIN lesions to prevent the development of invasive PDAC, as PDAC spreads rapidly outside the pancreas. This review aims to provide the latest knowledge on PanIN risk, pathology, cellular origin, genetic susceptibility, and diagnosis, while identifying research gaps that require further investigation in this understudied area of precancerous lesions. PanINs are classified into PanIN 1, PanIN 2, and PanIN 3, with PanIN 3 having the highest likelihood of developing into invasive PDAC. Differentiating between PanIN 2 and PanIN 3 is clinically significant. Genetic alterations found in PDAC are also present in PanIN and increase with the grade of PanIN. Imaging methods alone are insufficient for distinguishing PanIN, necessitating the use of genetic and molecular tests for identification. In addition, metabolomics technologies and miRNAs are playing an increasingly important role in the field of cancer diagnosis, offering more possibilities for efficient identification of PanIN. Although detecting and stratifying the risk of PanIN poses challenges, the combined utilization of imaging, genetics, and metabolomics holds promise for improving patient survival in this field.

Tumor recurrence after curative therapy and hepatocellular carcinoma (HCC) cells' resistance to conventional therapies is the reasons for the worse clinical results of HCC patients. A tiny population of cancer cells with a strong potential for self-renewal, differentiation, and tumorigenesis has been identified as cancer stem cells (CSCs). The discovery of CSC surface markers and the separation of CSC subpopulations from HCC cells have been made possible by recent developments in the study of hepatic (liver) CSCs. Hepatic CSC surface markers include epithelial cell adhesion molecules (EpCAM), CD133, CD90, CD13, CD44, OV-6, ALDH, and K19. CSCs have a significant influence on the development of cancer, invasiveness, self-renewal, metastasis, and drug resistance in HCC, and thus provide a therapeutic chance to treat HCC and avoid its recurrence. Therefore, it is essential to develop treatment approaches that specifically and effectively target hepatic stem cells. Given this, one potential treatment approach is to use particular small interfering RNA (siRNA) to target CSC, disrupting their behavior and microenvironment as well as changing their epigenetic state. The characteristics of CSCs in HCC are outlined in this study, along with new treatment approaches based on siRNA that may be used to target hepatic CSCs and overcome HCC resistance to traditional therapies.

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic and lethal disease. Gasdermins are primarily associated with necrosis via membrane permeabilization and pyroptosis, a lytic pro-inflammatory type of cell death. In this study, GSDMC upregulation during PDAC progression is reported. GSDMC directly induces genes related to stemness, EMT, and immune evasion. Targeting Gsdmc in murine PDAC models reprograms the immunosuppressive tumor microenvironment, rescuing the recruitment of anti-tumor immune cells through CXCL9. This not only results in diminished tumor initiation, growth and metastasis, but also enhances the response to KRASG12D inhibition and PD-1 checkpoint blockade, respectively. Mechanistically, it is discovered that ADAM17 cleaves GSDMC, releasing nuclear fragments binding to promoter regions of stemness, metastasis, and immune evasion-related genes. Pharmacological inhibition of GSDMC cleavage or prevention of its nuclear translocation is equally effective in suppressing GSDMC's downstream targets and inhibiting PDAC progression. The findings establish GSDMC as a potential therapeutic target for enhancing treatment response in this deadly disease.

Despite extensive research in chemotherapy, global cancer concerns persist, exacerbated by the challenge of drug resistance, which imposes economic and medical burdens. Natural compounds, particularly secondary metabolites from medicinal plants, present promising avenues for overcoming cancer drug resistance due to their diverse structures and essential pharmacological effects. This review provides a comprehensive exploration of cancer cell resistance mechanisms and target actions for reversing resistance and highlights the in vitro and in vivo efficacy of noteworthy alkaloids, flavonoids, and other compounds, emphasizing their potential as therapeutic agents. The molecular properties supporting ligand interactions are thoroughly examined, providing a robust theoretical foundation. The review concludes by discussing methods including quantitative structure-activity relationships and molecular docking, offering insights into screening potential candidates. Current trends in clinical treatment, contributing to a holistic understanding of the multifaceted approaches to address cancer drug resistance are also outlined.

Cancer stem cells (CSCs) are widely acknowledged as the drivers of tumor initiation, epithelial-mesenchymal transition (EMT) progression, and metastasis. Originating from both hematologic and solid malignancies, CSCs exhibit quiescence, pluripotency, and self-renewal akin to normal stem cells, thus orchestrating tumor heterogeneity and growth. Through a dynamic interplay with the tumor microenvironment (TME) and intricate signaling cascades, CSCs undergo transitions from differentiated cancer cells, culminating in therapy resistance and disease recurrence. This review undertakes an in-depth analysis of the multifaceted mechanisms underlying cancer stemness and CSC-mediated resistance to therapy. Intrinsic factors encompassing the TME, hypoxic conditions, and oxidative stress, alongside extrinsic processes such as drug efflux mechanisms, collectively contribute to therapeutic resistance. An exploration into key signaling pathways, including JAK/STAT, WNT, NOTCH, and HEDGEHOG, sheds light on their pivotal roles in sustaining CSCs phenotypes. Insights gleaned from preclinical and clinical studies hold promise in refining drug discovery efforts and optimizing therapeutic interventions, especially chimeric antigen receptor (CAR)-T cell therapy, cytokine-induced killer (CIK) cell therapy, natural killer (NK) cell-mediated CSC-targeting and others. Ultimately use of cell sorting and single cell sequencing approaches for elucidating the fundamental characteristics and resistance mechanisms inherent in CSCs will enhance our comprehension of CSC and intratumor heterogeneity, which ultimately would inform about tailored and personalized interventions.

Cancer stem cells (CSC) in colorectal cancer drive intratumoral heterogeneity and distant metastases. Previous research from our group showed that CSCs can be easily detected by autofluorescence (AF). The aim of the present study was to evaluate the potential role of AF CSCs as a prognostic biomarker for colorectal cancer relapse. Seventy-five freshly resected tumors were analyzed by flow cytometry. AF was categorized as high (H-AF) or low, and the results were correlated with histologic features [grade of differentiation, presence of metastases in lymph nodes (LN), perivascular and lymphovascular invasion] and clinical variables (time to relapse and overall survival). Nineteen of the 75 (25.3%) patients experienced relapse (local or distant); of these 19 patients, 13 showed positive LNs and 6 had H-AF. Of note, four of them died before 5 years. Although patients with H-AF CSC percentages in the global population experienced 1.5 times increased relapse [HR, 1.47; 95% confidence interval (0.60-3.63)], patients with H-AF CSC percentages and LN metastases had the highest risk of relapse [HR, 7.92; P < 0.004; 95% confidence interval (1.97-31.82)]. These data support AF as an accurate and feasible marker to identify CSCs in resected colorectal cancer. A strong statistical association between H-AF CSCs and the risk of relapse was observed, particularly in patients with positive LNs, suggesting that H-AF patients might benefit from adjuvant chemotherapy regimens and intensive surveillance due to their high propensity to experience disease recurrence. Significance: AF has been proven to be an accurate biomarker for CSC identification; however, to date, their role as a prognostic factor after resection of colorectal cancer tumors has not been investigated. Our results show that determining the presence of AF CSCs after tumor resection has prognostic value and represents a potentially important tool for the management of patients with colorectal cancer.

Flavins and their associated proteins have recently emerged as compelling players in the landscape of cancer biology. Flavins, encompassing flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), serve as coenzymes in a multitude of cellular processes, such as metabolism, apoptosis, and cell proliferation. Their involvement in oxidative phosphorylation, redox homeostasis, and enzymatic reactions has long been recognized. However, recent research has unveiled an extended role for flavins in the context of cancer. In parallel, riboflavin transporters (RFVTs), FAD synthase (FADS), and riboflavin kinase (RFK) have gained prominence in cancer research. These proteins, responsible for riboflavin uptake, FAD biosynthesis, and FMN generation, are integral components of the cellular machinery that governs flavin homeostasis. Dysregulation in the expression/function of these proteins has been associated with various cancers, underscoring their potential as diagnostic markers, therapeutic targets, and key determinants of cancer cell behavior. This review embarks on a comprehensive exploration of the multifaceted role of flavins and of the flavoproteins involved in nucleus-mitochondria crosstalk in cancer. We journey through the influence of flavins on cancer cell energetics, the modulation of RFVTs in malignant transformation, the diagnostic and prognostic significance of FADS, and the implications of RFK in drug resistance and apoptosis. This review also underscores the potential of these molecules and processes as targets for novel diagnostic and therapeutic strategies, offering new avenues for the battle against this relentless disease.

Cutaneous melanoma still represents a significant health burden worldwide, being responsible for the majority of skin cancer deaths. Key advances in therapeutic strategies have significantly improved patient outcomes; however, most patients experience drug resistance and tumor relapse. Cancer stem cells (CSCs) are a small subpopulation of cells in different tumors, including melanoma, endowed with distinctive capacities of self-renewal and differentiation into bulk tumor cells. Melanoma CSCs are characterized by the expression of specific biomarkers and intracellular pathways; moreover, they play a pivotal role in tumor onset, progression and drug resistance. In recent years, great efforts have been made to dissect the molecular mechanisms underlying the protumor activities of melanoma CSCs to provide the basis for novel CSC-targeted therapies. Herein, we highlight the intricate crosstalk between melanoma CSCs and bystander cells in the tumor microenvironment (TME), including immune cells, endothelial cells and cancer-associated fibroblasts (CAFs), and its role in melanoma progression. Specifically, we discuss the peculiar capacities of melanoma CSCs to escape the host immune surveillance, to recruit immunosuppressive cells and to educate immune cells toward an immunosuppressive and protumor phenotype. We also address currently investigated CSC-targeted strategies that could pave the way for new promising therapeutic approaches for melanoma care.

The novel Oxathiazinane derivative GP-2250 (Misetionamide) displays antineoplastic activity in vitro and in vivo, as previously shown in pancreatic cancer cells and in patient-derived mouse xenografts (PDX). Currently, GP 2250 is under phase I clinical trial in pancreatic ductal adenocarcinoma (PDAC). GP-2250 in combination with Gemcitabine displays a high synergistic capacity in various primary and established pancreatic cancer cell lines. Additionally, in the eight PDX models tested, the drug combination was superior in reducing tumor volume with an aggregate tumor regression (ATR) of 74% compared to Gemcitabine alone (ATR: 10%). Similarly, in a PDX maintenance setting following two weeks of treatment with nab-Paclitaxel plus Gemcitabine, the combination of GP-2250 plus Gemcitabine resulted in outstanding tumor control (ATR: 79%) compared to treatment with Gemcitabine alone (ATR: 19%). Furthermore, GP-2250 reduced the ratio of tumor-initiating CD133+ markers on the surface of PDAC cells in spheroid cultures, indicating a possible mechanism for the synergistic effect of both substances. Considering the high tolerability of GP 2250, these results may open up a new approach to maintenance therapy with GP-2250/Gemcitabine combination following nab-Paclitaxel plus Gemcitabine as first-line treatment.

An oxygen sensor-mounted fine-needle biopsy tool was used for in vivo measurement of oxygen levels in tumor xenografts. The system provides a means of measuring the oxygen content in harvested tumor tissue from specific locations. Oxygen in human tumor xenografts in a murine model was observed for over 1 min. Tissues were mapped in relation to oxygen tension (pO2) readings and sampled for conventional cytological examination. Careful modeling of the pO2 readings over 60 seconds yielded a diffusion coefficient for oxygen at the sensor tip, providing additional diagnostic information about the tissue before sampling. Oxygen level measurement may provide a useful adjunct to the use of biomarkers in tumor diagnosis.

Most high-risk neuroblastoma patients who relapse succumb to disease despite the existing therapy. We recently reported increased event-free and overall survival in neuroblastoma patients receiving difluoromethylornithine (DFMO) during maintenance therapy. The effect of DFMO on cellular processes associated with neuroblastoma tumorigenesis needs further elucidation. Previous studies have shown cytotoxicity with IC50 values >5-15 mM, these doses are physiologically unattainable in patients, prompting further mechanistic studies at therapeutic doses.

We characterized the effect of DFMO on cell viability, cell cycle, apoptosis, neurosphere formation, and protein expression in vitro using five established neuroblastoma cell lines (BE2C, CHLA-90, SHSY5Y, SMS-KCNR, and NGP) at clinically relevant doses of 0, 50, 100, 500, 1000, and 2500 μM. Limiting Dilution studies of tumor formation in murine models were performed. Statistical analysis was done using GraphPad and the level of significance set at p = 0.05.

There was not a significant loss of cell viability or gain of apoptotic activity in the in vitro assays (p > 0.05). DFMO treatment initiated G1 to S phase cell cycle arrest. There was a dose-dependent decrease in frequency and size of neurospheres and a dose-dependent increase in beta-galactosidase activity in all cell lines. Tumor formation was decreased in xenografts both with DFMO-pretreated cells and in mice treated with DFMO.

DFMO treatment is cytostatic at physiologically relevant doses and inhibits tumor initiation and progression in mice. This study suggests that DFMO, inhibits neuroblastoma by targeting cellular processes integral to neuroblastoma tumorigenesis at clinically relevant doses.

Optimal imaging strategies remain underdeveloped to maximize information for fluorescence microscopy while minimizing the harm to fragile living systems. Taking hint from the supercontinuum generation in ultrafast laser physics, we generated supercontinuum fluorescence from untreated unlabeled live samples before nonlinear photodamage onset. Our imaging achieved high-content cell phenotyping and tissue histology, identified bovine embryo polarization, quantified aging-related stress across cell types and species, demystified embryogenesis before and after implantation, sensed drug cytotoxicity in real-time, scanned brain area for targeted patching, optimized machine learning to track small moving organisms, induced two-photon phototropism of leaf chloroplasts under two-photon photosynthesis, unraveled microscopic origin of autumn colors, and interrogated intestinal microbiome. The results enable a facility-type microscope to freely explore vital molecular biology across life sciences.

Previous studies by our group have shown that oxidative phosphorylation (OXPHOS) is the main pathway by which pancreatic cancer stem cells (CSCs) meet their energetic requirements; therefore, OXPHOS represents an Achille's heel of these highly tumorigenic cells. Unfortunately, therapies that target OXPHOS in CSCs are lacking.

The safety and anti-CSC activity of a ruthenium complex featuring bipyridine and terpyridine ligands and one coordination labile position (Ru1) were evaluated across primary pancreatic cancer cultures and in vivo, using 8 patient-derived xenografts (PDXs). RNAseq analysis followed by mitochondria-specific molecular assays were used to determine the mechanism of action.

We show that Ru1 is capable of inhibiting CSC OXPHOS function in vitro, and more importantly, it presents excellent anti-cancer activity, with low toxicity, across a large panel of human pancreatic PDXs, as well as in colorectal cancer and osteosarcoma PDXs. Mechanistic studies suggest that this activity stems from Ru1 binding to the D-loop region of the mitochondrial DNA of CSCs, inhibiting OXPHOS complex-associated transcription, leading to reduced mitochondrial oxygen consumption, membrane potential, and ATP production, all of which are necessary for CSCs, which heavily depend on mitochondrial respiration.

Overall, the coordination complex Ru1 represents not only an exciting new anti-cancer agent, but also a molecular tool to dissect the role of OXPHOS in CSCs. Results indicating that the compound is safe, non-toxic and highly effective in vivo are extremely exciting, and have allowed us to uncover unprecedented mechanistic possibilities to fight different cancer types based on targeting CSC OXPHOS.

Sepsis is caused by a disordered host immune in response to infection and endothelial cells perform a crucial role in boosting immunity reaction in the pathophysiology of sepsis and septic organ failure. The aim of this study is to construct a novel erythrocyte membrane-derived nanosystems to reverse endothelial damage in sepsis. Herein, an innovative nanometer calcium metal-organic framework (Ca-MOF) is generated for the first time by using chelidonic acid as a ligand and calcium chloride as an ion donor for anti-inflammation. Then, zoliflodacin is loaded into Ca-MOF (CMZ) to sterilize and nanoscale erythrocyte membrane vesicles are prepared by modification with a γ3 peptide on the surface (γ3-RM) for precise targeting. Finally, γ3-RM camouflages the nanocore CMZ, to form novel erythrocyte membrane-camouflaged nanoparticle γ3-RCMZ. The superior performance of novel nanosystem results from its suitable biocompatibility, nontoxicity, specific targeting, and anti-inflammatory and bactericidal effects. Its anti-inflammatory mechanism mainly involves inhibiting the Caspase1-nuclear factor kappa-B (Caspase1-NF-κB) pathway and oxidative stress reduction to alleviate endothelial damage. Moreover, the findings have revealed for the first time that the bactericidal drug zoliflodacin also has anti-inflammatory effects in vivo and in vitro. Therefore, the novel nanosystem (γ3-RCMZ) provides a new nanotherapy strategy for sepsis treatment.

Pancreatic ductal adenocarcinoma (PDAC) is a profoundly aggressive and fatal cancer. One of the key factors defining its aggressiveness and resilience against chemotherapy is the existence of cancer stem cells (CSCs). The important task of discovering upstream regulators of stemness that are amenable for targeting in PDAC is essential for the advancement of more potent therapeutic approaches. In this study, we sought to elucidate the function of the nuclear receptor subfamily 5, group A, member 2 (NR5A2) in the context of pancreatic CSCs.

We modeled human PDAC using primary PDAC cells and CSC-enriched sphere cultures. NR5A2 was genetically silenced or inhibited with Cpd3. Assays included RNA-seq, sphere/colony formation, cell viability/toxicity, real-time PCR, western blot, immunofluorescence, ChIP, CUT&Tag, XF Analysis, lactate production, and in vivo tumorigenicity assays. PDAC models from 18 patients were treated with Cpd3-loaded nanocarriers.

Our findings demonstrate that NR5A2 plays a dual role in PDAC. In differentiated cancer cells, NR5A2 promotes cell proliferation by inhibiting CDKN1A. On the other hand, in the CSC population, NR5A2 enhances stemness by upregulating SOX2 through direct binding to its promotor/enhancer region. Additionally, NR5A2 suppresses MYC, leading to the activation of the mitochondrial biogenesis factor PPARGC1A and a shift in metabolism towards oxidative phosphorylation, which is a crucial feature of stemness in PDAC. Importantly, our study shows that the specific NR5A2 inhibitor, Cpd3, sensitizes a significant fraction of PDAC models derived from 18 patients to standard chemotherapy. This treatment approach results in durable remissions and long-term survival. Furthermore, we demonstrate that the expression levels of NR5A2/SOX2 can predict the response to treatment.

The findings of our study highlight the cell context-dependent effects of NR5A2 in PDAC. We have identified a novel pharmacological strategy to modulate SOX2 and MYC levels, which disrupts stemness and prevents relapse in this deadly disease. These insights provide valuable information for the development of targeted therapies for PDAC, offering new hope for improved patient outcomes. A Schematic illustration of the role of NR5A2 in cancer stem cells versus differentiated cancer cells, along with the action of the NR5A2 inhibitor Cpd3. B Overall survival of tumor-bearing mice following allocated treatment. A total of 18 PDX models were treated using a 2 x 1 x 1 approach (two animals per model per treatment); n=36 per group (illustration created with biorender.com ).

A large body of evidence indicates the existence of a complex pathophysiological relationship between cardiovascular diseases and cancer. Mitochondria are crucial organelles whose optimal activity is determined by quality control systems, which regulate critical cellular events, ranging from intermediary metabolism and calcium signaling to mitochondrial dynamics, cell death and mitophagy. Emerging data indicate that impaired mitochondrial quality control drives myocardial dysfunction occurring in several heart diseases, including cardiac hypertrophy, myocardial infarction, ischaemia/reperfusion damage and metabolic cardiomyopathies. On the other hand, diverse human cancers also dysregulate mitochondrial quality control to promote their initiation and progression, suggesting that modulating mitochondrial homeostasis may represent a promising therapeutic strategy both in cardiology and oncology. In this review, first we briefly introduce the physiological mechanisms underlying the mitochondrial quality control system, and then summarize the current understanding about the impact of dysregulated mitochondrial functions in cardiovascular diseases and cancer. We also discuss key mitochondrial mechanisms underlying the increased risk of cardiovascular complications secondary to the main current anticancer strategies, highlighting the potential of strategies aimed at alleviating mitochondrial impairment-related cardiac dysfunction and tumorigenesis. It is hoped that this summary can provide novel insights into precision medicine approaches to reduce cardiovascular and cancer morbidities and mortalities.

There is an urgent need for simple and non-invasive identification of live neural stem/progenitor cells (NSPCs) in the developing and adult brain as well as in disease, such as in brain tumors, due to the potential clinical importance in prognosis, diagnosis, and treatment of diseases of the nervous system. Here, we report a luminescent conjugated oligothiophene (LCO), named p-HTMI, for non-invasive and non-amplified real-time detection of live human patient-derived glioblastoma (GBM) stem cell-like cells and NSPCs. While p-HTMI stained only a small fraction of other cell types investigated, the mere addition of p-HTMI to the cell culture resulted in efficient detection of NSPCs or GBM cells from rodents and humans within minutes. p-HTMI is functionalized with a methylated imidazole moiety resembling the side chain of histidine/histamine, and non-methylated analogues were not functional. Cell sorting experiments of human GBM cells demonstrated that p-HTMI labeled the same cell population as CD271, a proposed marker for stem cell-like cells and rapidly migrating cells in glioblastoma. Our results suggest that the LCO p-HTMI is a versatile tool for immediate and selective detection of neural and glioma stem and progenitor cells.

This study introduces a rapid, volumetric live-cell imaging technique for visualizing autofluorescent sub-cellular structures and their dynamics by employing high-resolution Fourier light-field microscopy. We demonstrated this method by capturing lysosomal autofluorescence in fibroblasts and HeLa cells. Additionally, we conducted multicolor imaging to simultaneously observe lysosomal autofluorescence and fluorescently-labeled organelles such as lysosomes and mitochondria. We further analyzed the data to quantify the interactions between lysosomes and mitochondria. This research lays the foundation for future exploration of native cellular states and functions in three-dimensional environments, effectively reducing photodamage and eliminating the necessity for exogenous labels.

Pancreatic ductal adenocarcinoma (PDAC) is a type of cancer with a high mortality rate. Current treatments for PDACs often have side effects, and drug resistance in cancer stem cells (CSCs) would be also a problem. Cyclic guanosine monophosphate (cGMP) suppresses the mitochondrial function of PDACs and inhibits their CSC properties. Metabolic regulation plays a crucial role in the maintenance of CSC phenotype, and we hypothesized that cGMP induction suppresses cancer stem cell properties in the cancer cell through energy-related signaling pathways. We demonstrated that induction of cGMP upregulated the PPARα/PDK4 pathway and suppressed CSC properties in PDAC, and patients with pancreatic cancer with high PDK4 gene expression had a better prognosis than those with low gene expression. Therefore, these mechanisms may provide new therapeutic targets for the eradication of pancreatic CSCs.

Optical microscopes today have pushed the limits of speed, quality, and observable space in biological specimens revolutionizing how we view life today. Further, specific labeling of samples for imaging has provided insight into how life functions. This enabled label-based microscopy to percolate and integrate into mainstream life science research. However, the use of labelfree microscopy has been mostly limited, resulting in testing for bio-application but not bio-integration. To enable bio-integration, such microscopes need to be evaluated for their timeliness to answer biological questions uniquely and establish a long-term growth prospect. The article presents key label-free optical microscopes and discusses their integrative potential in life science research for the unperturbed analysis of biological samples.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive diseases with a poor 5-year survival rate. PDAC cells rely on various metabolic pathways to fuel their unlimited proliferation and metastasis. Reprogramming glucose, fatty acid, amino acid, and nucleic acid metabolisms contributes to PDAC cell growth. Cancer stem cells are the primary cell types that play a critical role in the progression and aggressiveness of PDAC. Emerging studies indicate that the cancer stem cells in PDAC tumors are heterogeneous and show specific metabolic dependencies. In addition, understanding specific metabolic signatures and factors that regulate these metabolic alterations in the cancer stem cells of PDAC paves the way for developing novel therapeutic strategies targeting CSCs. In this review, we discuss the current understanding of PDAC metabolism by specifically exploring the metabolic dependencies of cancer stem cells. We also review the current knowledge of targeting these metabolic factors that regulate CSC maintenance and PDAC progression.

The term "cancer stem cells" (CSCs) refers to cancer cells that exhibit traits parallel to normal stem cells, namely the potential to give rise to every type of cell identified in a tumor microenvironment. It has been found that CSCs usually develops from other neoplastic cells or non-cancerous somatic cells by acquiring stemness and malignant characteristics through particular genetic modifications. A trivial number of CSCs, identified in solid and liquid cancer, can give rise to an entire tumor population with aggressive anticancer drug resistance, metastasis, and invasiveness. Besides, cancer stem cells manipulate their intrinsic and extrinsic features, regulate the metabolic pattern of the cell, adjust efflux-influx efficiency, modulate different signaling pathways, block apoptotic signals, and cause genetic and epigenetic alterations to retain their pluripotency and ability of self-renewal. Notably, to keep the cancer stem cells' ability to become malignant cells, mesenchymal stem cells, tumor-associated fibroblasts, immune cells, etc., interact with one another. Furthermore, CSCs are characterized by the expression of particular molecular markers that carry significant diagnostic and prognostic significance. Because of this, scientific research on CSCs is becoming increasingly imperative, intending to understand the traits and behavior of cancer stem cells and create more potent anticancer therapeutics to fight cancer at the CSC level. In this review, we aimed to elucidate the critical role of CSCs in the onset and spread of cancer and the characteristics of CSCs that promote severe resistance to targeted therapy.

Specific targets for cancer treatment are highly desirable, but still remain to be discovered. While previous reports suggested that CAPRIN-1 localizes in the cytoplasm, here we now show that part of this molecule is strongly expressed on the cell membrane surface in most solid cancers, but not normal tissues. Notably, the membrane expression of CAPRIN-1 extended to the subset of highly tumorigenic cancer stem cells and epithelial-mesenchymal transition (EMT)-induced metastatic cancer cells. In addition, we revealed that cancer cells with particularly high CAPRIN-1 surface expression exhibited enhanced tumorigenicity. We generated a therapeutic humanized anti-CAPRIN-1 antibody (TRK-950), which strongly and specifically binds to various cancer cells and shows antitumor effects via engagement of immune cells. TRK-950 was further developed as a new cancer drug and a series of preclinical studies demonstrates its therapeutic potency in tumor-bearing mouse models and safety in a relevant cynomolgus monkey model. Together, our data demonstrate that CAPRIN-1 is a novel and universal target for cancer therapies. A phase I clinical study of TRK-950 has been completed (NCT02990481) and a phase Ib study (combination with approved drugs) is currently underway (NCT03872947) in the United States and France. In parallel, a phase I study in Japan is in progress as well (NCT05423262).

Antibody-based cancer therapies have been demonstrated to be effective, but are only approved for a limited number of targets, because the majority of these markers is shared with healthy tissue, which may result in adverse effects. Here, we have successfully identified CAPRIN-1 as a novel truly cancer-specific target, universally expressed on membranes of various cancer cells including cancer stem cells. Clinical studies are underway for the anti-CAPRIN-1 therapeutic antibody TRK-950.

Experimental results indicate that riboflavin is involved in tumorigenesis. Data regarding the relationship between riboflavin and colorectal cancer (CRC) are limited, and findings vary between observational studies.

This was a case-control retrospective study.

This study aimed to evaluate the associations between serum riboflavin level and sporadic CRC risk.

In total, 389 participants were enrolled in this study - including 83 CRC patients without family history and 306 healthy controls - between January 2020 and March 2021 at the Department of Colorectal Surgery and Endoscope Center at Xinhua Hospital, Shanghai Jiao Tong University School of Medicine. Age, sex, body mass index, history of polyps, disease conditions (e.g., diabetes), medications, and eight other vitamins were used as confounding factors. Adjusted smoothing spline plots, subgroup analysis, and multivariate logistic regression analysis were conducted to estimate the relative risk between serum riboflavin levels and sporadic CRC risk. After fully adjusting for the confounding factors, an increased risk of colorectal cancer was suggested for individuals with higher levels of serum riboflavin (OR = 1.08 (1.01, 1.15), p = 0.03) in a dose-response relationship.

Our results support the hypothesis that higher levels of riboflavin may play a role in facilitating colorectal carcinogenesis. The finding of high levels of circulating riboflavin in patients with CRC warrants further investigation.

The distribution of fluorescence signals measured with flow cytometry can be influenced by several factors, including qualitative and quantitative properties of the used fluorochromes, optical properties of the detection system, as well as the variability within the analyzed cell population itself. Most of the single cell samples prepared from in vitrocultures or clinical specimens contain a variable cell cycle component. Cell cycle, together with changes in the cell size, are two of the factors that alter the functional properties of analyzed cells and thus affect the interpretation of obtained results. Here, we describe the association between cell cycle status and cell size, and the variability in the distribution of fluorescence intensity as determined with flow cytometry, at population scale. We show that variability in the distribution of background and specific fluorescence signals is related to the cell cycle state of the selected population, with the 10% low fluorescence signal fraction enriched mainly in cells in their G0/G1 cell cycle phase, and the 10% high fraction containing cells mostly in the G2/M phase. Therefore we advise using caution and additional experimental validation when comparing populations defined by fractions at both ends of fluorescence signal distribution to avoid biases caused by the effect of cell cycle and cell size.

The resistance of cancer cells to chemotherapeutic drugs greatly reduces the therapeutic effect in cancer patients, and the toxic side effects caused by chemotherapy also seriously affect the quality of life of patients. The combination of epigallocatechin-3-gallate (EGCG), the main active ingredient in tea, with cisplatin, 5-FU, doxorubicin and paclitaxel enhances their sensitizing effect on tumors and combats the drug resistance of cancer cells. These effects seem to be mediated by a variety of mechanisms, including combating drug resistance mediated by cancer stem cells, enhancing drug sensitivity, inducing cell cycle arrest and apoptosis, and blocking angiogenesis. In addition, EGCG can suppress a series of adverse effects caused by chemotherapy, such as gastrointestinal disorders, nephrotoxicity and cardiotoxicity, through its anti-inflammatory and antioxidant effects and improve the quality of life of patients. However, the low bioavailability and off-target effects of EGCG and its reactivity with some chemotherapeutic agents limit its clinical application. The nanomodification of EGCG and chemotherapeutic drugs not only enhances the antitumor activity but also prolongs the survival time of tumor-bearing mice, and has the advantage of low toxicity. Therefore, this review aims to discuss the current status and challenges regarding the use of EGCG in combination with chemotherapy drugs in the treatment of cancer. In general, EGCG is a promising adjuvant for chemotherapy.

A series of oxidized cysteinyl peptides ([P-Cys-X-OMe]₂; P = Boc or H; X = Trp or Glu) showed vesicular and fibrillar assemblies. The anatomy of the self-assembled vesicles from the water-soluble cystine peptide [Cys-Trp-OMe]₂ (1a) has been investigated by using various fluorescent probes such as ammonium 8-anilinonaphthalene-1-sulfonate, Nile Red and pyrene. The morphological characterization was carried out by fluorescence lifetime imaging microscopy (FLIM) and super resolution-structured illumination microscopy (SR-SIM) utilizing the autofluorescence of the vesicles stemming from the self-assembly. The self-assembled structures are also observed in solution as evident from the quantitative phase images obtained using a dual-mode digital holographic microscope (DHM) system. Present investigations show that the self-assembly is enthalpy- and entropy-driven in the aqueous medium. Based on the CD spectral studies, we proposed that 1a organizes into vesicles through the sequestration of indole units. We observed that the solutions of dipeptides 1a-b exhibit autofluorescence in the blue region upon excitation at a wavelength >350 nm. Detailed spectroscopic studies on the peptides lacking tryptophan 2a-b unequivocally showed that the autofluorescence stems exclusively from peptide aggregation. Our experimental results with appropriate controls revealed that the clustering of carbonyl chromophores is central to autofluorescence. Autofluorescence was also used to probe the vesicle formation without using any external fluorescent probe. To the best of our knowledge, this is the first report on autofluorescent vesicles formed by the spontaneous association of dipeptides. We also found that the vesicles formed by 1a can act as a host for guests like C₆₀. The biocompatibility and biodegradability of these peptides along with the autofluorescent nature and guest binding ability of peptide-based vesicles offer numerous applications in the biomedical area.

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive disease characterized by its metastatic potential and chemoresistance. These traits are partially attributable to the highly tumorigenic pancreatic cancer stem cells (PaCSCs). Interestingly, these cells show unique features in order to sustain their identity and functionality, some of them amenable for therapeutic intervention. Screening of phospho-receptor tyrosine kinases revealed that PaCSCs harbored increased activation of anaplastic lymphoma kinase (ALK). We subsequently demonstrated that oncogenic ALK signaling contributes to tumorigenicity in PDAC patient-derived xenografts (PDXs) by promoting stemness through ligand-dependent activation. Indeed, the ALK ligands midkine (MDK) or pleiotrophin (PTN) increased self-renewal, clonogenicity and CSC frequency in several in vitro local and metastatic PDX models. Conversely, treatment with the clinically-approved ALK inhibitors Crizotinib and Ensartinib decreased PaCSC content and functionality in vitro and in vivo, by inducing cell death. Strikingly, ALK inhibitors sensitized chemoresistant PaCSCs to Gemcitabine, as the most used chemotherapeutic agent for PDAC treatment. Consequently, ALK inhibition delayed tumor relapse after chemotherapy in vivo by effectively decreasing the content of PaCSCs. In summary, our results demonstrate that targeting the MDK/PTN-ALK axis with clinically-approved inhibitors impairs in vivo tumorigenicity and chemoresistance in PDAC suggesting a new treatment approach to improve the long-term survival of PDAC patients.

Conventional techniques for in vitro cancer drug screening require labor-intensive formalin fixation, paraffin embedding, and dye staining of tumor tissues at fixed endpoints. This way of assessment discards the valuable pharmacodynamic information in live cells over time. Here, we found endogenous lipofuscin-like autofluorescence acutely accumulated in the cell death process. Its unique red autofluorescence could report the apoptosis without labeling and continuously monitor the treatment responses in 3D tumor-culture models. Lifetime imaging of lipofuscin-like red autofluorescence could further distinguish necrosis from apoptosis of cells. Moreover, this endogenous fluorescent marker could visualize the apoptosis in live zebrafish embryos during development. Overall, this study validates that lipofuscin-like autofluorophore is a generic cell death marker. Its characteristic autofluorescence could label-free predict the efficacy of anti-cancer drugs in organoids or animal models.

The appearance of chemoresistance in cancer is a major issue. The main barriers to conventional tumor chemotherapy are undesirable toxic effects and multidrug resistance. Cancer nanotherapeutics were developed to get around the drawbacks of conventional chemotherapy. Through clinical evaluation of thoughtfully developed nano delivery systems, cancer nanotherapeutics have recently offered unmatched potential to comprehend and combat drug resistance and toxicity. In different design approaches, including passive targeting, active targeting, nanomedicine, and multimodal nanomedicine combination therapy, were successful in treating cancer in this situation. Even though cancer nanotherapy has achieved considerable technological development, tumor biology complexity and heterogeneity and a lack of full knowledge of nano-bio interactions remain important hurdles to future clinical translation and commercialization. The recent developments and advancements in cancer nanotherapeutics utilizing a wide variety of nanomaterial-based platforms to overcome cancer treatment resistance are covered in this article. Additionally, an evaluation of different nanotherapeutics-based approaches to cancer treatment, such as tumor microenvironment targeted techniques, sophisticated delivery methods for the precise targeting of cancer stem cells, as well as an update on clinical studies are discussed. Lastly, the potential for cancer nanotherapeutics to overcome tumor relapse and the therapeutic effects and targeted efficacies of modern nanosystems are analyzed.

Intratumour heterogeneity (ITH) has become an important focus of cancer research in recent years. ITH describes the cellular variation that enables tumour evolution, including tumour progression, metastasis and resistance to treatment. The selection and expansion of genetically distinct treatment-resistant cancer cell clones provides one explanation for treatment failure. However, tumour cell variation need not be genetically encoded. In pancreatic ductal adenocarcinoma (PDAC) in particular, the complex tumour microenvironment as well as crosstalk between tumour and stromal cells result in exceptionally variable tumour cell phenotypes that are also highly adaptable. In this review we discuss four different types of phenotypic heterogeneity within PDAC, from morphological to metabolic heterogeneity. We suggest that these different types of ITH are not independent, but, rather, can inform one another. Lastly, we highlight recent findings that suggest how therapeutic efforts may halt PDAC progression by constraining cellular heterogeneity.

Pancreatic cancer (PC) has one of the highest fatality rates and the currently available therapeutic options are not sufficient to improve its overall poor prognosis. In addition to insufficient effectiveness of anticancer treatments, the lack of clear early symptoms and early metastatic spread maintain the PC survival rates at a low level. Metabolic reprogramming is among the hallmarks of cancer and could be exploited for the diagnosis and treatment of PC. PC is characterized by its heterogeneity and, apart from molecular subtypes, the identification of metabolic subtypes in PC could aid in the development of more individualized therapeutic approaches and may lead to improved clinical outcomes. In addition to the deregulated utilization of glucose in aerobic glycolysis, PC cells can use a wide range of substrates, including branched‑chain amino acids, glutamine and lipids to fulfil their energy requirements, as well as biosynthetic needs. The tumor microenvironment in PC supports tumor growth, metastatic spread, treatment resistance and the suppression of the host immune response. Moreover, reciprocal interactions between cancer and stromal cells enhance their metabolic reprogramming. PC stem cells (PCSCs) with an increased resistance and distinct metabolic properties are associated with disease relapses and cancer spread, and represent another significant candidate for therapeutic targeting. The present review discusses the metabolic signatures observed in PC, a disease with a multifaceted and often transient metabolic landscape. In addition, the metabolic pathways utilized by PC cells, as well as stromal cells are discussed, providing examples of how they could present novel targets for therapeutic interventions and elaborating on how interactions between the various cell types affect their metabolism. Furthermore, the importance of PCSCs is discussed, focusing specifically on their metabolic adaptations.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and the adoptive immunotherapy of which is worth studying. CD133, a kind of cancer stem cell (CSC) antigen, together with glypican-3 (GPC3) has been proved to be highly expressed in HCC cells and both of them are used as targets to generate chimeric antigen receptor (CAR) T cells. But there are limitations like "off-target" toxicity, low transfection efficacy and weak antitumor ability in CAR T cells treatment.

The peripheral blood was acquired from healthy donors and T cells were separated by density-gradient centrifugation. We used an electroporation system to deliver anti-CD133 and anti-GPC3 single chain Fragment variable (scFv) structures as target genes into the T cells. The cell membrane was opened by the momentary electric current effect, and the target gene was delivered into the cell by non-viral minicircle DNA (mcDNA) vector. The flow cytometry and western blot assays were used to detect whether the two scFv were simultaneously transfected and the transfection efficacy of this bispecific CAR T cell generation method. We respectively detected the in vitro and in vivo tumor-suppression efficacy of CAR T cells through the CCK-8 assays and the HCC xenograft mice models. The CoG133-CAR T cells containing both CD133 and GPC3 antigen recognition sites were the effector cells. CD133-CAR T cells and GPC3-CAR T cells were defined as single-targeted control groups, normal T and mock T cells were defined as blank control groups.

The mcDNA vector accommodated two target gene structures successfully transfected to generate bispecific CAR T cells. The detection methods on gene level and protein level confirmed that CoG133-CAR T cells had considerable transfection efficiency and exhibited both antigen-binding capacity of CD133 and GPC3. Compared to single-targeted CAR T cells or control T cells, CoG133-CAR T cells performed enhanced eliminated efficacy against CD133 and GPC3 double-positive HCC cell line in vitro and HCC xenograft mice in vivo. Hematoxylin and eosin (H&E) staining indicated no fatal "off-target" combination existed on CoG133-CAR T cells and major organs.

Our study suggests that it is with higher efficiency and more safety to prepare bispecific CAR T cells through non-viral mcDNA vectors. CoG133-CAR T cells have enhanced tumor-suppression capacity through dual antigen recognition and internal activation. It provides an innovative strategy for CAR T therapy of HCC, even solid tumors.