Standardized Digital Image Analysis of PD-L1 Expression in Head and Neck Squamous Cell Carcinoma Reveals Intra- and Inter-Sample Heterogeneity with Therapeutic Implications.

For practical reasons, in many studies PD-L1 expression is measured by combined positive score (CPS) from a single tumor sample. This does not reflect the heterogeneity of PD-L1 expression in head and neck squamous cell carcinoma (HNSCC). We investigated the extent and relevance of PD-L1 expression heterogeneity in HNSCC analyzing primary tumors and recurrences (LRs), as well as metastases. Tumor tissue from 200 HNSCC patients was immunohistochemically stained for PD-L1 and analyzed using image-analysis software QuPath v3.4 with multiple specimens per patient. CPS was ≥20 in 25.6% of primary tumors. Intra-tumoral heterogeneity led to a therapeutically relevant underestimation of PD-L1 expression in 28.7% of patients, when only one specimen per patient was analyzed. Inter-tumoral differences in PD-L1 expression between primary tumors and lymph node metastasis (LNM) or LR occurred in 44.4% and 61.5% (CPS) and in 40.6% and 50% of cases (TPS). Overall survival was increased in patients with CPS ≥ 1 vs. CPS < 1 in primary tumors and LNM (hazard ratio: 0.46 and 0.35; p < 0.005); CPS in LR was not prognostic. Our analysis shows clinically relevant intra- and inter-sample heterogeneity of PD-L1 expression in HNSCC. To account for heterogeneity and improve patient selection for immunotherapy, multiple sample analyses should be performed, particularly in patients with CPS/TPS < 1.

Revolution in Cancer Treatment: How Are Intelligently Designed Nanostructures Changing the Game?

Nanoparticles (NPs) are extremely important tools to overcome the limitations imposed by therapeutic agents and effectively overcome biological barriers. Smart designed/tuned nanostructures can be extremely effective for cancer treatment. The selection and design of nanostructures and the adjustment of size and surface properties are extremely important, especially for some precision treatments and drug delivery (DD). By designing specific methods, an important era can be opened in the biomedical field for personalized and precise treatment. Here, we focus on advances in the selection and design of nanostructures, as well as on how the structure and shape, size, charge, and surface properties of nanostructures in biological fluids (BFs) can be affected. We discussed the applications of specialized nanostructures in the therapy of head and neck cancer (HNC), which is a difficult and aggressive type of cancer to treat, to give an impetus for novel treatment approaches in this field. We also comprehensively touched on the shortcomings, current trends, and future perspectives when using nanostructures in the treatment of cancer.

Biosynthesized tumor acidity and MMP dual-responsive plant toxin gelonin for robust cancer therapy.

Among all kinds of anticancer agents, small molecule drugs produce an unsatisfactory therapeutic effect due to the lack of selectivity, notorious drug resistance and side effects. Therefore, researchers have begun to pay extensive attention to macromolecular drugs with high efficacy and specificity. As a plant toxin, gelonin exerts potent antitumor activity via inhibiting intracellular protein synthesis. However, gelonin lacks a translocation domain, and thus its poor cellular uptake leads to low outcomes of antitumor response. Here, tumor acidity and matrix metalloproteinase (MMP) dual-responsive functional gelonin (Trx-PVGLIG-pHLIP-gelonin, TPpG), composed of a thioredoxin (Trx) tag, a pH low insertion peptide (pHLIP), an MMP-responsive motif PVGLIG hexapeptide and gelonin, was innovatively proposed and biologically synthesized by a gene recombination technique. TPpG exhibited good thermal and serum stability, showed MMP responsiveness and could enter tumor cells under weakly acidic conditions, especially for MMP2-overexpressing HT1080 cells. Compared to low MMP2-expressing MCF-7 cells, TPpG displayed enhanced in vitro antitumor efficacy to HT1080 cells at pH 6.5 as determined by different methods. Likewise, TPpG was much more effective in triggering cell apoptosis and inhibiting protein synthesis in HT1080 cells than in MCF-7 cells. Intriguingly, with enhanced stability and pH/MMP dual responsiveness, TPpG notably inhibited subcutaneous HT1080 xenograft growth in mice and no noticeable off-target side effect was observed. This ingeniously designed strategy aims at providing new perspectives for the development of a smart platform that can intelligently respond to a tumor microenvironment for efficient protein delivery.

The Magic Triangle in Oral Potentially Malignant Disorders: Vitamin D, Vitamin D Receptor, and Malignancy.

OPMDs (oral potentially malignant disorders) are a group of disorders affecting the oral mucosa that are characterized by aberrant cell proliferation and a higher risk of malignant transformation. Vitamin D (VitD) and its receptor (VDR) have been extensively studied for their potential contributions to the prevention and therapeutic management of various diseases and neoplastic conditions, including oral cancer. Observational studies suggest correlations between VitD deficiency and higher cancer risk, worse prognosis, and increased mortality rates. Interestingly, emerging data also suggest a link between VitD insufficiency and the onset or progression of OPMDs. Understanding the role of the VitD-VDR axis not only in established oral tumors but also in OPMDs might thus enable early detection and prevention of malignant transformation. With this article, we want to provide an overview of current knowledge about OPMDs and VitD and investigate their potential association and ramifications for clinical management of OPMDs.

Exosomes in Cancer Progression and Therapy Resistance: Molecular Insights and Therapeutic Opportunities.

The development of therapy resistance still represents a major hurdle in treating cancers, leading to impaired treatment success and increased patient morbidity. The establishment of minimally invasive liquid biopsies is a promising approach to improving the early diagnosis, as well as therapy monitoring, of solid tumors. Because of their manifold functions in the tumor microenvironment, tumor-associated small extracellular vesicles, referred to as exosomes, have become a subject of intense research. Besides their important roles in cancer progression, metastasis, and the immune response, it has been proposed that exosomes also contribute to the acquisition and transfer of therapy resistance, mainly by delivering functional proteins and RNAs, as well as facilitating the export of active drugs or functioning as extracellular decoys. Extensive research has focused on understanding the molecular mechanisms underlying the occurrence of resistance and translating these into strategies for early detection. With this review, we want to provide an overview of the current knowledge about the (patho-)biology of exosomes, as well as state-of-the-art methods of isolation and analysis. Furthermore, we highlight the role of exosomes in tumorigenesis and cancer treatment, where they can function as therapeutic agents, biomarkers, and/or targets. By focusing on their roles in therapy resistance, we will reveal new paths of exploiting exosomes for cancer diagnosis and treatment.

Tuning Nanobodies' Bioactivity: Coupling to Ultrasmall Gold Nanoparticles Allows the Intracellular Interference with Survivin.

Nanobodies are highly affine binders, often used to track disease-relevant proteins inside cells. However, they often fail to interfere with pathobiological functions, required for their clinical exploitation. Here, a nanobody targeting the disease-relevant apoptosis inhibitor and mitosis regulator Survivin (SuN) is utilized. Survivin's multifaceted functions are regulated by an interplay of dynamic cellular localization, dimerization, and protein-protein interactions. However, as Survivin harbors no classical "druggable" binding pocket, one must aim at blocking extended protein surface areas. Comprehensive experimental evidence demonstrates that intracellular expression of SuN allows to track Survivin at low nanomolar concentrations but failed to inhibit its biological functions. Small angle X-ray scattering of the Survivin-SuN complex locates the proposed interaction interface between the C-terminus and the globular domain, as such not blocking any pivotal interaction. By clicking multiple SuN to ultrasmall (2 nm) gold nanoparticles (SuN-N), not only intracellular uptake is enabled, but additionally, Survivin crosslinking and interference with mitotic progression in living cells are also enabled. In sum, it is demonstrated that coupling of nanobodies to nanosized scaffolds can be universally applicable to improve their function and therapeutic applicability.

Targeting STING Activation by Antigen-Inspired MnO2 Nanovaccines Optimizes Tumor Radiotherapy.

Immune checkpoint blockers therapy can improve the radiotherapy-induced immunosuppression by enhancing interferon secretion, but still suffer from low clinical response rate and potential adverse effects. Mn2+ -mediated activation of interferon gene stimulator (STING) pathway provides an alternative for combination radioimmunotherapy of tumor. However, it is still a challenge for specific delivery of Mn2+ to innate immune cells and targeting activation of STING pathway. Herein, a novel antigen-inspired MnO2 nanovaccine is fabricated as Mn2+ source and functionalized with mannose, enabling it to target innate immune cells to activate the STING pathway. Meanwhile, the release of Mn2+ in the intracellular lysosomes can also be for magnetic resonance imaging to monitor the dynamic distribution of nanovaccines in vivo. The targeting activation of STING pathway can enhance radiotherapy-induced immune responses for inhibiting local and distant tumors, and resisting tumor metastasis. The study proposes an optimized radiotherapy strategy through targeting STING activation of antigen-inspired nanovaccines.

Complex Structures Made Simple - Continuous Flow Production of Core Cross-Linked Polymeric Micelles for Paclitaxel Pro-Drug-Delivery.

Translating innovative nanomaterials to medical products requires efficient manufacturing techniques that enable large-scale high-throughput synthesis with high reproducibility. Drug carriers in medicine embrace a complex subset of tasks calling for multifunctionality. Here, the synthesisof pro-drug-loaded core cross-linked polymeric micelles (CCPMs) in a continuous flow processis reported, which combines the commonly separated steps of micelle formation, core cross-linking, functionalization, and purification into a single process. Redox-responsive CCPMs are formed from thiol-reactive polypept(o)ides of polysarcosine-block-poly(S-ethylsulfonyl-l-cysteine) and functional cross-linkers based on dihydrolipoic acid hydrazide for pH-dependent release of paclitaxel. The precisely controlled microfluidic process allows the production of spherical micelles (Dh  = 35 nm) with low polydispersity values (PDI < 0.1) while avoiding toxic organic solvents and additives with unfavorable safety profiles. Self-assembly and cross-linking via slit interdigital micromixers produces 350-700 mg of CCPMs/h per single system, while purification by online tangential flow filtration successfully removes impurities (unimer ≤ 0.5%). The formed paclitaxel-loaded CCPMs possess the desired pH-responsive release profile, display stable drug encapsulation, an improved toxicity profile compared to Abraxane (a trademark of Bristol-Myers Squibb), and therapeutic efficiency in the B16F1-xenotransplanted zebrafish model. The combination of reactive polymers, functional cross-linkers, and microfluidics enables the continuous-flow synthesis of therapeutically active CCPMs in a single process.

Exploiting Vitamin D Receptor and Its Ligands to Target Squamous Cell Carcinomas of the Head and Neck.

Vitamin D (VitD) and its receptor (VDR) have been intensively investigated in many cancers. As knowledge for head and neck cancer (HNC) is limited, we investigated the (pre)clinical and therapeutic relevance of the VDR/VitD-axis. We found that VDR was differentially expressed in HNC tumors, correlating to the patients' clinical parameters. Poorly differentiated tumors showed high VDR and Ki67 expression, whereas the VDR and Ki67 levels decreased from moderate to well-differentiated tumors. The VitD serum levels were lowest in patients with poorly differentiated cancers (4.1 ± 0.5 ng/mL), increasing from moderate (7.3 ± 4.3 ng/mL) to well-differentiated (13.2 ± 3.4 ng/mL) tumors. Notably, females showed higher VitD insufficiency compared to males, correlating with poor differentiation of the tumor. To mechanistically uncover VDR/VitD's pathophysiological relevance, we demonstrated that VitD induced VDR nuclear-translocation (VitD < 100 nM) in HNC cells. RNA sequencing and heat map analysis showed that various nuclear receptors were differentially expressed in cisplatin-resistant versus sensitive HNC cells including VDR and the VDR interaction partner retinoic acid receptor (RXR). However, RXR expression was not significantly correlated with the clinical parameters, and cotreatment with its ligand, retinoic acid, did not enhance the killing by cisplatin. Moreover, the Chou-Talalay algorithm uncovered that VitD/cisplatin combinations synergistically killed tumor cells (VitD < 100 nM) and also inhibited the PI3K/Akt/mTOR pathway. Importantly, these findings were confirmed in 3D-tumor-spheroid models mimicking the patients' tumor microarchitecture. Here, VitD already affected the 3D-tumor-spheroid formation, which was not seen in the 2D-cultures. We conclude that novel VDR/VitD-targeted drug combinations and nuclear receptors should also be intensely explored for HNC. Gender-specific VDR/VitD-effects may be correlated to socioeconomic differences and need to be considered during VitD (supplementation)-therapies.

Taspase1 Facilitates Topoisomerase IIß-Mediated DNA Double-Strand Breaks Driving Estrogen-Induced Transcription.

The human protease Taspase1 plays a pivotal role in developmental processes and cancerous diseases by processing critical regulators, such as the leukemia proto-oncoprotein MLL. Despite almost two decades of intense research, Taspase1's biology is, however, still poorly understood, and so far its cellular function was not assigned to a superordinate biological pathway or a specific signaling cascade. Our data, gained by methods such as co-immunoprecipitation, LC-MS/MS and Topoisomerase II DNA cleavage assays, now functionally link Taspase1 and hormone-induced, Topoisomerase IIß-mediated transient DNA double-strand breaks, leading to active transcription. The specific interaction with Topoisomerase IIα enhances the formation of DNA double-strand breaks that are a key prerequisite for stimulus-driven gene transcription. Moreover, Taspase1 alters the H3K4 epigenetic signature upon estrogen-stimulation by cleaving the chromatin-modifying enzyme MLL. As estrogen-driven transcription and MLL-derived epigenetic labelling are reduced upon Taspase1 siRNA-mediated knockdown, we finally characterize Taspase1 as a multifunctional co-activator of estrogen-stimulated transcription.

Impact of Peptide Sequences on Their Structure and Function: Mimicking of Virus-Like Nanoparticles for Nucleic Acid Delivery.

We rationally designed a series of amphiphilic hepta-peptides enriched with a chemically conjugated guanidiniocarbonylpyrrole (GCP) unit at the lysine side chain. All peptides are composed of polar (GCP) and non-polar (cyclohexyl alanine) residues but differ in their sequence periodicity, resulting in different secondary as well as supramolecular structures. CD spectra revealed the assembly of ß-sheet-, α-helical and random structures for peptides 1, 2 and 3, respectively. Consequently, this enabled the formation of distinct supramolecular assemblies such as fibres, nanorod-like or spherical aggregates. Notably, all three cationic peptides are equipped with the anion-binding GCP unit and thus possess a nucleic acid-binding centre. However, only the helical (2) and the unstructured (3) peptide were able to assemble into small virus-like DNA-polyplexes and effectively deliver DNA into cells. Notably, as both peptides (2 and 3) were also capable of siRNA-delivery, they could be utilized to downregulate expression of the caner-relevant protein Survivin.

Dual activity inhibition of threonine aspartase 1 by a single bisphosphate ligand.

Therapy resistance remains a challenge for the clinics. Here, dual-active chemicals that simultaneously inhibit independent functions in disease-relevant proteins are desired though highly challenging. As a model, we here addressed the unique protease threonine aspartase 1, involved in various cancers. We hypothesized that targeting basic residues in its bipartite nuclear localization signal (NLS) by precise bisphosphate ligands inhibits additional steps required for protease activity. We report the bisphosphate anionic bivalent inhibitor 11d, selectively binding to the basic NLS cluster (220KKRR223) with high affinity (K D = 300 nM), thereby disrupting its interaction and function with Importin α (IC50 = 6 µM). Cell-free assays revealed that 11d additionally affected the protease's catalytic substrate trans-cleavage activity. Importantly, functional assays comprehensively demonstrated that 11d inhibited threonine aspartase 1 also in living tumor cells. We demonstrate for the first time that intracellular interference with independent key functions in a disease-relevant protein by an inhibitor binding to a single site is possible.

The Vitamin D Receptor-BIM Axis Overcomes Cisplatin Resistance in Head and Neck Cancer.

Treatment success of head and neck squamous cell carcinoma (HNSCC) is often hindered by cisplatin resistance. As inherent and acquired therapy resistance counteracts improvement in long-term survival, novel multi-targeting strategies triggering cancer cell apoptosis are urgently required. Here, we identify the vitamin D receptor (VDR) as being significantly overexpressed in tumors of HNSCC patients (n = 604; p = 0.0059), correlating with tumor differentiation (p = 0.0002), HPV status (p = 0.00026), and perineural invasion (p = 0.0087). The VDR, a member of the nuclear receptor superfamily, is activated by its ligand vitamin D (VitD) and analogs, triggering multiple cellular responses. As we found that the VDR was also upregulated in our cisplatin-resistant HNSCC models, we investigated its effect on overcoming cisplatin resistance. We discovered that VitD/cisplatin combinations synergistically killed even cisplatin-resistant cells at clinically achievable levels. Similar results were obtained for the clinically used VitD analog Maxacalcitol. Moreover, VitD/cisplatin combinations inhibited tumor cell migration by E-cadherin upregulation. Signaling pathway analyses revealed that VitD co-treatments triggered cancer cell death by increasing the expression of the pro-apoptotic BCL-2 family protein BIM. BIM's pro-apoptotic activity in HNSCC cells was confirmed by ectopic overexpression studies. Importantly, BIM expression is positively associated with HNSCC patients' (n = 539) prognosis, as high expression correlated with improved survival (p = 0.0111), improved therapy response (p = 0.0026), and remission (p = 0.004). Collectively, by identifying, for the first time, the VDR/BIM axis, we here provide a molecular rationale for the reported anti-cancer activity of VitD/analogs in combination therapies. Our data also suggest its exploitation as a potential strategy to overcome cisplatin resistance in HNSCC and other malignancies by inducing additional pro-apoptotic pathways.

Functionalized Ultrasmall Iron Oxide Nanoparticles for T1-Weighted Magnetic Resonance Imaging of Tumor Hypoxia.

Hypoxia is a common biological condition in many malignant solid tumors that plays an imperative role in regulating tumor growth and impacting the treatment's therapeutic effect. Therefore, the hypoxia assessment is of great significance in predicting tumor development and evaluating its prognosis. Among the plenty of existing tumor diagnosis techniques, magnetic resonance imaging (MRI) offers certain distinctive features, such as being free of ionizing radiation and providing images with a high spatial resolution. In this study, we develop a fluorescent traceable and hypoxia-sensitive T1-weighted MRI probe (Fe3O4-Met-Cy5.5) via conjugating notable hypoxia-sensitive metronidazole moiety and Cy5.5 dye with ultrasmall iron oxide (Fe3O4) nanoparticles. The results of in vitro and in vivo experiments show that Fe3O4-Met-Cy5.5 has excellent performance in relaxivity, biocompatibility, and hypoxia specificity. More importantly, the obvious signal enhancement in hypoxic areas indicates that the probe has great feasibility for sensing tumor hypoxia via T1-weighted MRI. These promising results may unlock the potential of Fe3O4 nanoparticles as T1-weighted contrast agents for the development of clinical hypoxia probes.

Are Routine Chest X-rays Necessary following Thoracic Surgery? A Systematic Literature Review and Meta-Analysis.

(1) Background: The number of chest X-rays that are performed in the perioperative window of thoracic surgery varies. Many clinics X-ray patients daily, while others only perform X-rays if there are clinical concerns. The purpose of this study was to assess the evidence of perioperative X-rays following thoracic surgery and estimate the clinical value with regard to changes in patient care. (2) Methods: A systematic literature research was conducted up until November 2021. Studies reporting X-ray outcomes in adult patients undergoing general thoracic surgery were included. (3) Results: In total, 11 studies (3841 patients/4784 X-rays) were included. The X-ray resulted in changes in patient care in 488 cases (10.74%). In patients undergoing mediastinoscopic lymphadenectomy or thoracoscopic sympathectomy, postoperative X-ray never led to changes in patient care. (4) Conclusions: There are no data to recommend an X-ray before surgery or to recommend daily X-rays. X-rays immediately after surgery seem to rarely have any consequences. It is probably reasonable to keep requesting X-rays after drain removal since they serve multiple purposes and alter patient care in 7.30% of the cases.

Impact of Secretion-Active Osteoblast-Specific Factor 2 in Promoting Progression and Metastasis of Head and Neck Cancer.

Treatment success of head and neck cancer (HNC) is still hampered by tumor relapse due to metastases. Our study aimed to identify biomarkers by exploiting transcriptomics profiles of patient-matched metastases, primary tumors, and normal tissue mucosa as well as the TCGA HNC cohort data sets. Analyses identified osteoblast-specific factor 2 (OSF-2) as significantly overexpressed in lymph node metastases and primary tumors compared to normal tissue. High OSF-2 levels correlate with metastatic disease and reduced overall survival of predominantly HPV-negative HNC patients. No significant correlation was observed with tumor localization or therapy response. These findings were supported by the fact that OSF-2 expression was not elevated in cisplatin-resistant HNC cell lines. OSF-2 was strongly expressed in tumor-associated fibroblasts, suggesting a tumor microenvironment-promoting function. Molecular cloning and expression studies of OSF-2 variants from patients identified an evolutionary conserved bona fide protein secretion signal (1MIPFLPMFSLLLLLIVNPINA21). OSF-2 enhanced cell migration and cellular survival under stress conditions, which could be mimicked by the extracellular administration of recombinant protein. Here, OSF-2 executes its functions via ß1 integrin, resulting in the phosphorylation of PI3K and activation of the Akt/PKB signaling pathway. Collectively, we suggest OSF-2 as a potential prognostic biomarker and drug target, promoting metastases by supporting the tumor microenvironment and lymph node metastases survival rather than by enhancing primary tumor proliferation or therapy resistance.

Molecularly engineered tumor acidity-responsive plant toxin gelonin for safe and efficient cancer therapy.

Due to the unsatisfactory therapeutic efficacy and inexorable side effects of small molecule antineoplastic agents, extensive efforts have been devoted to the development of more potent macromolecular agents with high specificity. Gelonin is a plant-derived protein toxin that exhibits robust antitumor effect via inactivating ribosomes and inhibiting protein synthesis. Nonetheless, its poor internalization ability to tumor cells has compromised the therapeutic promise of gelonin. In this study, a tumor acidity-responsive intracellular protein delivery system ─ functional gelonin (Trx-pHLIP-Gelonin, TpG) composed of a thioredoxin (Trx) tag, a pH low insertion peptide (pHLIP) and gelonin, was designed and obtained by genetic recombination technique for the first time. TpG could effectively enter into tumor cells under weakly acidic conditions and markedly suppress tumor cell proliferation via triggering cell apoptosis and inhibiting protein synthesis. Most importantly, treatment by intravenous injection into subcutaneous SKOV3 solid tumors in a mouse model showed that TpG was much more effective than gelonin in curtailing tumor growth rates with negligible toxicity. Collectively, our present work suggests that the tumor acidity-targeted delivery manner endowed by pHLIP offers a new avenue for efficient delivery of other bioactive substances to acidic diseased tissues.

Identification of cytokeratin24 as a tumor suppressor for the management of head and neck cancer.

To improve management of head and neck squamous cell carcinoma patients, we need to increase our understanding of carcinogenesis, to identify biomarkers, and drug targets. This study aimed to identify novel biomarkers by providing transcriptomics profiles of matched primary tumors, lymph node metastasis, and non-malignant tissue of 20 HNSCC patients as well as by bioinformatic analyses of a TCGA HNSCC cohort, comprising 554 patients. We provide cancer cell signaling networks differentially expressed in tumors versus metastases, such as mesenchymal-epithelial transition, and structural integrity networks. As a proof of principle study, we exploited the data sets and performed functional analyses of a novel cytokeratin, cytokeratin24 (cKRT24), which had not been described as biomarker for tumors before. Survival analysis revealed that low cKRT24 expression correlated with poor overall survival in HNSCC. Experimentally, downregulation of cKRT24 in primary tumors, metastases, and HNSCC cell lines was verified on mRNA and protein level. Cloning and ectopic overexpression of cKRT24 not only affected viability and growth of HNSSC cell lines, but also inhibited tumor growth in murine xenograft studies. We conclude that cKRT24 functions as a tumor suppressor in HNSCC, and may serve as an additional prognostic biomarker and novel target to support current HNSCC treatments.

Targeting Cancer Chemotherapy Resistance by Precision Medicine-Driven Nanoparticle-Formulated Cisplatin.

Therapy resistance is the major cause of cancer death. As patients respond heterogeneously, precision/personalized medicine needs to be considered, including the application of nanoparticles (NPs). The success of therapeutic NPs requires to first identify clinically relevant resistance mechanisms and to define key players, followed by a rational design of biocompatible NPs capable to target resistance. Consequently, we employed a tiered experimental pipeline from in silico to analytical and in vitro to overcome cisplatin resistance. First, we generated cisplatin-resistant cancer cells and used next-generation sequencing together with CRISPR/Cas9 knockout technology to identify the ion channel LRRC8A as a critical component for cisplatin resistance. LRRC8A's cisplatin-specificity was verified by testing free as well as nanoformulated paclitaxel or doxorubicin. The clinical relevance of LRRC8A was demonstrated by its differential expression in a cohort of 500 head and neck cancer patients, correlating with patient survival under cisplatin therapy. To overcome LRRC8A-mediated cisplatin resistance, we constructed cisplatin-loaded, polysarcosine-based core cross-linked polymeric NPs (NPCis, Ø âˆ¼ 28 nm) with good colloidal stability, biocompatibility (low immunogenicity, low toxicity, prolonged in vivo circulation, no complement activation, no plasma protein aggregation), and low corona formation properties. 2D/3D-spheroid cell models were employed to demonstrate that, in contrast to standard of care cisplatin, NPCis significantly (p < 0.001) eradicated all cisplatin-resistant cells by circumventing the LRRC8A-transport pathway via the endocytic delivery route. We here identified LRRC8A as critical for cisplatin resistance and suggest LRRC8A-guided patient stratification for ongoing or prospective clinical studies assessing therapy resistance to nanoscale platinum drug nanoformulations versus current standard of care formulations.

IsoMAG-An Automated System for the Immunomagnetic Isolation of Squamous Cell Carcinoma-Derived Circulating Tumor Cells.

BACKGROUND: detailed information about circulating tumor cells (CTCs) as an indicator of therapy response and cancer metastasis is crucial not only for basic research but also for diagnostics and therapeutic approaches. Here, we showcase a newly developed IsoMAG IMS system with an optimized protocol for fully automated immunomagnetic enrichment of CTCs, also revealing rare CTC subpopulations. METHODS: using different squamous cell carcinoma cell lines, we developed an isolation protocol exploiting highly efficient EpCAM-targeting magnetic beads for automated CTC enrichment by the IsoMAG IMS system. By FACS analysis, we analyzed white blood contamination usually preventing further downstream analysis of enriched cells. RESULTS: 1 µm magnetic beads with tosyl-activated hydrophobic surface properties were found to be optimal for automated CTC enrichment. More than 86.5% and 95% of spiked cancer cells were recovered from both cell culture media or human blood employing our developed protocol. In addition, contamination with white blood cells was minimized to about 1200 cells starting from 7.5 mL blood. Finally, we showed that the system is applicable for HNSCC patient samples and characterized isolated CTCs by immunostaining using a panel of tumor markers. CONCLUSION: Herein, we demonstrate that the IsoMAG system allows the detection and isolation of CTCs from HNSCC patient blood for disease monitoring in a fully-automated process with a significant leukocyte count reduction. Future developments seek to integrate the IsoMAG IMS system into an automated microfluidic-based isolation workflow to further facilitate single CTC detection also in clinical routine.