Creation of an Isogenic H/N/KRAS-Less Mouse Embryonic Fibroblast Cell Line Panel Derived from a Size-Sorted Diploid Clone.

Cell line panels have proven to be an invaluable tool for investigators researching a range of topics from drug mechanism or drug sensitivity studies to disease-specific etiology. The cell lines used in these panels may range from heterogeneous tumor populations grown from primary tumor isolations to genetically engineered clonal cell lines which express specific gene isoforms. Mouse embryonic fibroblast (MEF) cells are a commonly used cell line for biological research due to their accessibility and ease of genetic manipulation. This chapter will describe the process of creating a size-sorted diploid (SSDC) clonal cell panel expressing specific RAS isoforms from a previously engineered RAS-less MEF cell line pool.

Cell States in Cancer: Drivers, Passengers, and Trailers.

SUMMARY: Cancer is traditionally perceived through a genetic lens, with therapeutic strategies targeting oncogenic driver mutations. We advocate an overarching framework recognizing tumors as comprising driver, passenger, and trailer cell states: Tailoring therapies to simultaneously target driver genetics and cell states may enhance effectiveness and durability. SIGNIFICANCE: We redefine cancer progression by introducing a model that categorizes tumor cells into "driver," "passenger," and "trailer" phenotypes, expanding the focus on genetic aberrations to cellular behavior. This approach offers a roadmap to guide refining therapeutic strategies for more precise and durable cancer treatments that address tumor heterogeneity and plasticity.

The Cancer Spectrum Theory.

SUMMARY: Biological characteristics of tumors are heterogeneous, forming spectra in terms of several factors such as age at onset, anatomic spatial localization, tumor subtyping, and the degree of tumor aggressiveness (encompassing a neoplastic property spectrum). Instead of blindly using dichotomized approaches, the application of the multicategorical and continuous analysis approaches to detailed cancer spectrum data can contribute to a better understanding of the etiology of cancer, ultimately leading to effective prevention and precision oncology. We provide examples of cancer spectra and emphasize the importance of integrating the cancer spectrum theory into large-scale population cancer research.

Comparative Preclinical Evaluation of HYNIC-Modified Designed Ankyrin Repeat Proteins G3 for the 99mTc-Based Imaging of HER2-Expressing Malignant Tumors.

HER2 status determination is a necessary step for the proper choice of therapy and selection of patients for the targeted treatment of cancer. Targeted radiotracers such as radiolabeled DARPins provide a noninvasive and effective way for the molecular imaging of HER2 expression. This study aimed to evaluate tumor-targeting properties of three 99mTc-labeled DARPin G3 variants containing Gly-Gly-Gly-Cys (G3C), (Gly-Gly-Gly-Ser)3-Cys ((G3S)3C), or Glu-Glu-Glu-Cys (E3C) amino acid linkers at the C-terminus and conjugated to the HYNIC chelating agent, as well as to compare them with the clinically evaluated DARPin G3 labeled with 99mTc(CO)3 using the (HE)3-tag at the N-terminus. The labeling of DARPin G3-HYNIC variants provided radiochemical yields in the range of 50-80%. Labeled variants bound specifically to human HER2-expressing cancer cell lines with affinities in the range of 0.5-3 nM. There was no substantial influence of the linker and HYNIC chelator on the binding of 99mTc-labeled DARPin G3 variants to HER2 in vitro; however, [99mTc]Tc-G3-(G3S)3C-HYNIC had the highest affinity. Comparative biodistribution of [99mTc]Tc-G3-G3C-HYNIC, [99mTc]Tc-G3-(G3S)3C-HYNIC, [99mTc]Tc-G3-E3C-HYNIC, and [99mTc]Tc-(HE)3-G3 in healthy CD1 mice showed that there was a strong influence of the linkers on uptake in normal tissues. [99mTc]Tc-G3-E3C-HYNIC had an increased retention of activity in the liver and the majority of other organs compared to the other conjugates. The tumor uptake of [99mTc]Tc-G3-(G3S)3C-HYNIC and [99mTc]Tc-(HE)3-G3 in Nu/j mice bearing SKOV-3 xenografts was similar. The specificity of tumor targeting in vivo was demonstrated for both tracers. [99mTc]Tc-G3-(G3S)3C-HYNIC provided comparable, although slightly lower tumor-to-lung, tumor-to spleen and tumor-to-liver ratios than [99mTc]Tc-(HE)3-G3. Radiolabeling of DARPin G3-HYNIC conjugates with 99mTc provided the advantage of a single-step radiolabeling procedure; however, the studied HYNIC conjugates did not improve imaging contrast compared to the 99mTc-tricarbonyl-labeled DARPin G3. At this stage, [99mTc]Tc-(HE)3-G3 remains the most promising candidate for the clinical imaging of HER2-overexpressing cancers.

Stressed out neutrophils drive metastasis.

Stress hormones can contribute to cancer progression, but how immune cells play a role in this process is unclear. In a recent study in Cancer Cell, He et al. showed that glucocorticoids potentiate metastasis by skewing neutrophils toward pro-tumorigenic functions.

Exosomal microRNAs as potential biomarkers and therapeutic targets in corneal diseases.

Exosomes are a subtype of extracellular vesicle (EV) that are released and found in almost all body fluids. Exosomes consist of and carry a variety of bioactive molecules, including genetic information in the form of microRNAs (miRNAs). miRNA, a type of small non-coding RNA, plays a key role in regulating genes by suppressing their translation. miRNAs are often disrupted in the pathophysiology of different conditions, including eye disease. The stability and easy detectability of exosomal miRNAs in body fluids make them promising biomarkers for the diagnosis of different diseases. Additionally, due to the natural delivery capabilities of exosomes, they can be modified to transport therapeutic miRNAs to specific recipient cells. Most exosome research has primarily focused on cancer, so there is limited research highlighting the importance of exosomes in ocular biology, particularly in cornea-associated pathologies. This review provides an overview of the existing evidence regarding the primary functions of exosomal miRNAs and their potential role in diagnostic and therapeutic applications in the human cornea.

Tumor microenvironment restricts IL-10 induced multipotent progenitors to myeloid-lymphatic phenotype.

Lymphangiogenesis is induced by local pro-lymphatic growth factors and bone marrow (BM)-derived myeloid-lymphatic endothelial cell progenitors (M-LECP). We previously showed that M-LECP play a significant role in lymphangiogenesis and lymph node metastasis in clinical breast cancer (BC) and experimental BC models. We also showed that differentiation of mouse and human M-LECP can be induced through sequential activation of colony stimulating factor-1 (CSF-1) and Toll-like receptor-4 (TLR4) pathways. This treatment activates the autocrine interleukin-10 (IL-10) pathway that, in turn, induces myeloid immunosuppressive M2 phenotype along with lymphatic-specific proteins. Because IL-10 is implicated in differentiation of numerous lineages, we sought to determine whether this pathway specifically promotes the lymphatic phenotype or multipotent progenitors that can give rise to M-LECP among other lineages. Analyses of BM cells activated either by CSF-1/TLR4 ligands in vitro or orthotopic breast tumors in vivo showed expansion of stem/progenitor population and coincident upregulation of markers for at least four lineages including M2-macrophage, lymphatic endothelial, erythroid, and T-cells. Induction of cell plasticity and multipotency was IL-10 dependent as indicated by significant reduction of stem cell markers and those for multiple lineages in differentiated cells treated with anti-IL-10 receptor (IL-10R) antibody or derived from IL-10R knockout mice. However, multipotent CD11b+/Lyve-1+/Ter-119+/CD3e+ progenitors detected in BM appeared to split into a predominant myeloid-lymphatic fraction and minor subsets expressing erythroid and T-cell markers upon establishing tumor residence. Each sub-population was detected at a distinct intratumoral site. This study provides direct evidence for differences in maturation status between the BM progenitors and those reaching tumor destination. The study results suggest preferential tumor bias towards expansion of myeloid-lymphatic cells while underscoring the role of IL-10 in early BM production of multipotent progenitors that give rise to both hematopoietic and endothelial lineages.

Isolation and Single-Cell Transcriptomic Analysis of Murine Regulatory B Cells.

Regulatory B (Breg) cells have been demonstrated to play an important role in the inhibition of a wide range of immunological responses, and they are absent or malfunction in autoimmune diseases like lupus. Breg cells can control immunological responses and keep the immune system in a balanced state by releasing immunosuppressive cytokines such as transforming growth factor-beta (TGF-ß) and interleukin-10 (IL-10), which in turn promote regulatory T (Treg) cells and reduce effector T cell responses. Breg cells have also been linked to the modulation of cancer immunity. Due to their immunosuppressive role, in the context of cancer, Breg cells aid in tumor immune evasion and promote tumor progression. Nonetheless, it has been established that Breg cells are involved in both cancer immunity and autoimmunity, and their characterizations beyond surface markers, for example, on the transcriptomic level, are essential for our understanding of Breg biology in health and disease. In this chapter, using lupus-prone MRL/lpr mice, we describe a Breg cell isolation protocol for the purpose of single-cell RNA sequencing analysis.

Oncostatin M signaling drives cancer-associated skeletal muscle wasting.

Progressive weakness and muscle loss are associated with multiple chronic conditions, including muscular dystrophy and cancer. Cancer-associated cachexia, characterized by dramatic weight loss and fatigue, leads to reduced quality of life and poor survival. Inflammatory cytokines have been implicated in muscle atrophy; however, available anticytokine therapies failed to prevent muscle wasting in cancer patients. Here, we show that oncostatin M (OSM) is a potent inducer of muscle atrophy. OSM triggers cellular atrophy in primary myotubes using the JAK/STAT3 pathway. Identification of OSM targets by RNA sequencing reveals the induction of various muscle atrophy-related genes, including Atrogin1. OSM overexpression in mice causes muscle wasting, whereas muscle-specific deletion of the OSM receptor (OSMR) and the neutralization of circulating OSM preserves muscle mass and function in tumor-bearing mice. Our results indicate that activated OSM/OSMR signaling drives muscle atrophy, and the therapeutic targeting of this pathway may be useful in preventing muscle wasting.

Integrating multiplexed imaging and multiscale modeling identifies tumor phenotype conversion as a critical component of therapeutic T cell efficacy.

Cancer progression is a complex process involving interactions that unfold across molecular, cellular, and tissue scales. These multiscale interactions have been difficult to measure and to simulate. Here, we integrated CODEX multiplexed tissue imaging with multiscale modeling software to model key action points that influence the outcome of T cell therapies with cancer. The initial phenotype of therapeutic T cells influences the ability of T cells to convert tumor cells to an inflammatory, anti-proliferative phenotype. This T cell phenotype could be preserved by structural reprogramming to facilitate continual tumor phenotype conversion and killing. One takeaway is that controlling the rate of cancer phenotype conversion is critical for control of tumor growth. The results suggest new design criteria and patient selection metrics for T cell therapies, call for a rethinking of T cell therapeutic implementation, and provide a foundation for synergistically integrating multiplexed imaging data with multiscale modeling of the cancer-immune interface. A record of this paper's transparent peer review process is included in the supplemental information.

Advances in Engineered Macrophages: A New Frontier in Cancer Immunotherapy.

Macrophages, as pivotal cells within the tumour microenvironment, significantly influence the impact of and reactions to treatments for solid tumours. The rapid evolution of bioengineering technology has revealed the vast potential of engineered macrophages in immunotherapy, disease diagnosis, and tissue engineering. Given this landscape, the goal of harnessing and innovating macrophages as a novel strategy for solid tumour immunotherapy cannot be overstated. The diverse strategies for engineered macrophages in the realm of cancer immunotherapy, encompassing macrophage drug delivery systems, chimeric antigen receptor macrophage therapy, and synergistic treatment approaches involving bacterial outer membrane vesicles and macrophages, are meticulously examined in this review. These methodologies are designed to enhance the therapeutic efficacy of macrophages against solid tumours, particularly those that are drug-resistant and metastatic. Collectively, these immunotherapies are poised to supplement and refine current solid tumour treatment paradigms, thus heralding a new frontier in the fight against malignant tumours.

The senescence journey in cancer immunoediting.

Cancer progression is continuously controlled by the immune system which can identify and destroy nascent tumor cells or inhibit metastatic spreading. However, the immune system and its deregulated activity in the tumor microenvironment can also promote tumor progression favoring the outgrowth of cancers capable of escaping immune control, in a process termed cancer immunoediting. This process, which has been classified into three phases, i.e. "elimination", "equilibrium" and "escape", is influenced by several cancer- and microenvironment-dependent factors. Senescence is a cellular program primed by cells in response to different pathophysiological stimuli, which is based on long-lasting cell cycle arrest and the secretion of numerous bioactive and inflammatory molecules. Because of this, cellular senescence is a potent immunomodulatory factor promptly recruiting immune cells and actively promoting tissue remodeling. In the context of cancer, these functions can lead to both cancer immunosurveillance and immunosuppression. In this review, the authors will discuss the role of senescence in cancer immunoediting, highlighting its context- and timing-dependent effects on the different three phases, describing how senescent cells promote immune cell recruitment for cancer cell elimination or sustain tumor microenvironment inflammation for immune escape. A potential contribution of senescent cells in cancer dormancy, as a mechanism of therapy resistance and cancer relapse, will be discussed with the final objective to unravel the immunotherapeutic implications of senescence modulation in cancer.

A phase I open-label, dose-escalation study of NUC-3373, a targeted thymidylate synthase inhibitor, in patients with advanced cancer (NuTide:301).

PURPOSE: 5-fluorouracil (5-FU) is inefficiently converted to the active anti-cancer metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP), is associated with dose-limiting toxicities and challenging administration schedules. NUC-3373 is a phosphoramidate nucleotide analog of fluorodeoxyuridine (FUDR) designed to overcome these limitations and replace fluoropyrimidines such as 5-FU. PATIENTS AND METHODS: NUC-3373 was administered as monotherapy to patients with advanced solid tumors refractory to standard therapy via intravenous infusion either on Days 1, 8, 15 and 22 (Part 1) or on Days 1 and 15 (Part 2) of 28-day cycles until disease progression or unacceptable toxicity. Primary objectives were maximum tolerated dose (MTD) and recommended Phase II dose (RP2D) and schedule of NUC-3373. Secondary objectives included pharmacokinetics (PK), and anti-tumor activity. RESULTS: Fifty-nine patients received weekly NUC-3373 in 9 cohorts in Part 1 (n = 43) and 3 alternate-weekly dosing cohorts in Part 2 (n = 16). They had received a median of 3 prior lines of treatment (range: 0-11) and 74% were exposed to prior fluoropyrimidines. Four experienced dose-limiting toxicities: two Grade (G) 3 transaminitis; one G2 headache; and one G3 transient hypotension. Commonest treatment-related G3 adverse event of raised transaminases occurred in < 10% of patients. NUC-3373 showed a favorable PK profile, with dose-proportionality and a prolonged half-life compared to 5-FU. A best overall response of stable disease was observed, with prolonged progression-free survival. CONCLUSION: NUC-3373 was well-tolerated in a heavily pre-treated solid tumor patient population, including those who had relapsed on prior 5-FU. The MTD and RP2D was defined as 2500 mg/m2 NUC-3373 weekly. NUC-3373 is currently in combination treatment studies. TRIAL REGISTRATION: Clinicaltrials.gov registry number NCT02723240. Trial registered on 8th December 2015. https://clinicaltrials.gov/study/NCT02723240 .

Tumor-activated in situ synthesis of single-atom catalysts for O2-independent photodynamic therapy based on water-splitting.

Single-atom catalysts (SACs) have attracted interest in photodynamic therapy (PDT), while they are normally limited by the side effects on normal tissues and the interference from the Tumor Microenvironment (TME). Here we show a TME-activated in situ synthesis of SACs for efficient tumor-specific water-based PDT. Upon reduction by upregulated GSH in TME, C3N4-Mn SACs are obtained in TME with Mn atomically coordinated into the cavity of C3N4 nanosheets. This in situ synthesis overcomes toxicity from random distribution and catalyst release in healthy tissues. Based on the Ligand-to-Metal charge transfer (LMCT) process, C3N4-Mn SACs exhibit enhanced absorption in the red-light region. Thereby, a water-splitting process is induced by C3N4-Mn SACs under 660 nm irradiation, which initiates the O2-independent generation of highly toxic hydroxyl radical (·OH) for cancer-specific PDT. Subsequently, the ·OH-initiated lipid peroxidation process is demonstrated to devote effective cancer cell death. The in situ synthesized SACs facilitate the precise cancer-specific conversion of inert H2O to reactive ·OH, which facilitates efficient cancer therapy in female mice. This strategy achieves efficient and precise cancer therapy, not only avoiding the side effects on normal tissues but also overcoming tumor hypoxia.

Ferroptosis is an effective strategy for cancer therapy.

Ferroptosis is a form of intracellular iron-dependent cell death that differs from necrosis, autophagy and apoptosis. Intracellular iron mediates Fenton reaction resulting in lipid peroxidation production, which in turn promotes cell death. Although cancer cell exhibit's ability to escape ferroptosis by multiple pathways such as SLC7A11, GPX4, induction of ferroptosis could inhibit cancer cell proliferation, migration and invasion. In tumor microenvironment, ferroptosis could affect immune cell (T cells, macrophages etc.) activity, which in turn regulates tumor immune escape. In addition, ferroptosis in cancer cells could activate immune cell activity by antigen processing and presentation. Therefore, ferroptosis could be an effective strategy for cancer therapy such as chemotherapy, radiotherapy, and immunotherapy. In this paper, we reviewed the role of ferroptosis on tumor progression and therapy, which may provide a strategy for cancer treatment.

[Molecular Pathology Diagnosis as Companion Diagnostics].

As genomic medicine advances, opportunities for molecular pathology diagnosis by pathologists to be used as companion diagnostics is increasing. Pathological specimens must be useful not only for pathological diagnosis, but also for genetic testing panel and molecular pathology diagnosis. Companion diagnostics performed by pathologists uses immunohistochemical staining and fluorescence in situ hybridization to determine patient eligibility for molecular target drugs and immune checkpoint inhibitors. By accurately observing a wide variety of diagnostic criteria and performing with high precision, pathological diagnosis will become closer to therapeutic pathology.

First-in-human study of GFH018, a small molecule inhibitor of transforming growth factor-ß receptor I inhibitor, in patients with advanced solid tumors.

BACKGROUND: Transforming growth factor-ß (TGF-ß) is a cytokine with multiple functions, including cell growth regulation, extracellular matrix production, angiogenesis homeostasis adjustment and et al. TGF-ß pathway activation promotes tumor metastasis/progression and mediates epithelial-mesenchymal transmission suppressing immunosurveillance in advanced tumors. GFH018, a small molecule inhibitor blocking TGF-ß signal transduction, inhibits the progression and/or metastasis of advanced cancers. This first-in-human study evaluated the safety, tolerability, pharmacokinetics (PK), and efficacy of GFH018 monotherapy in patients with advanced solid tumors. METHODS: This phase I, open-label, multicenter study used a modified 3+3 dose escalation and expansion design. Adult patients with advanced solid tumors failing the standard of care were enrolled. Starting at 5 mg, eight dose levels up to 85 mg were evaluated. Patients received GFH018 BID (14d-on/14d-off) starting on the 4th day after a single dose on cycle 1, day 1. Subsequent cycles were defined as 28 days. The study also explored the safety of 85 mg BID 7d-on/7d-off. Adverse events were graded using NCI criteria for adverse events (NCI-CTCAE v5.0). PK was analyzed using a noncompartmental method. Efficacy was evaluated using RECIST 1.1. Blood samples were collected for biomarker analysis. RESULTS: Fifty patients were enrolled and received at least one dose of GFH018. No dose-limiting toxicity occurred, and the maximum tolerated dose was not reached. Forty-three patients (86.0%) had at least one treatment-related adverse event (TRAE), and three patients (6.0%) had ≥ G3 TRAEs. The most common TRAEs (any grade/grade ≥3) were AST increased (18%/0%), proteinuria (14%/2%), anemia (14%/2%), and ALT increased (12%/0%). No significant cardiotoxicity or bleeding was observed. GFH018 PK was linear and dose-independent, with a mean half-life of 2.25-8.60 h from 5 - 85 mg. Nine patients (18.0%) achieved stable disease, and one patient with thymic carcinoma achieved tumor shrinkage, with the maximum target lesion decreased by 18.4%. Serum TGF-ß1 levels were not associated with clinical responses. The comprehensive recommended dose for Phase II was defined as 85 mg BID 14d-on/14d-off. CONCLUSIONS: GFH018 monotherapy presented a favorable safety profile without cardiac toxicity or bleeding. Modest efficacy warrants further studies, including combination strategies. TRIAL REGISTRATION: ClinicalTrial. gov ( https://www. CLINICALTRIALS: gov/ ), NCT05051241. Registered on 2021-09-02.

[Comparative pathology in oncology-Best practice]. / Vergleichende Pathologie in der onkologischen Forschung.

Comparative experimental pathology is a research field at the interface of human and veterinary medicine. It is focused on the comparative study of similarities and differences between spontaneous and experimentally induced diseases in animals (animal models) compared to human diseases. The use of animal models for studying human diseases is an essential component of biomedical research. Interdisciplinary teams with species-specific expertise should collaborate wherever possible and maintain close communication. Mutual openness, cooperation, and willingness to learn form the basis for a fruitful collaboration. Research projects jointly led by or involving both animal and human pathologists make a significant contribution to high-quality biomedical research. Such approaches are promising not only in oncological research, as outlined in this article, but also in other research areas where animal models are regularly used, such as infectiology, neurology, and developmental biology.

Quantitative performance assessment of Ultivue multiplex panels in formalin-fixed, paraffin-embedded human and murine tumor specimens.

We present a rigorous validation strategy to evaluate the performance of Ultivue multiplex immunofluorescence panels. We have quantified the accuracy and precision of four different multiplex panels (three human and one mouse) in tumor specimens with varying levels of T cell density. Our results show that Ultivue panels are typically accurate wherein the relative difference in cell proportion between a multiplex image and a 1-plex image is less than 20% for a given biomarker. Ultivue panels exhibited relatively high intra-run precision (CV ≤ 25%) and relatively low inter-run precision (CV >> 25%) which can be remedied by using local intensity thresholding to gate biomarker positivity. We also evaluated the reproducibility of cell-cell distance estimates measured from multiplex images which show high intra- and inter-run precision. We introduce a new metric, multiplex labeling efficiency, which can be used to benchmark the overall fidelity of the multiplex data across multiple batch runs. Taken together our results provide a comprehensive characterization of Ultivue panels and offer practical guidelines for analyzing multiplex images.