BACKGROUND:Photodynamic therapy (PDT) uses light to generate reactive oxygen species (ROS) that initiate cytotoxic responses in cancer cells. Photoimmunotherapy (PIT) improves PDT specificity by linking photosensitizers (PS) to monoclonal antibodies. A promising monoclonal antibody (mAb) for immunoconjugates is atezolizumab (Tecentriq), an EMA/FDA-approved PD-L1 immune checkpoint inhibitor for treating non-small cell lung cancer (NSCLC). OBJECTIVE:Our goal was to: Optimize the cytotoxic efficacy of a photoimmunoconjugate of eosin-5-isothiocyanate and atezolizumab (EITC-atezolizumab) in NSCLC cells; Study the uptake and intracellular transport of atezolizumab; Evaluate EITC-atezolizumab as a candidate for photochemical internalization (PCI) of the ribosome-inactivating protein gelonin. METHODS:Cytotoxic efficacy of EITC-atezolizumab was evaluated in NSCLC cell lines H1975 (PD-L1-high) and A549 (PD-L1-low) using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Confocal microscopy was employed to observe the uptake and intracellular localization of AlexaFluor647-atezolizumab and its colocalization with either LysoTracker™Blue or an anti-CD63 antibody labelled with Alexa488. RESULTS:EITC-atezolizumab PIT significantly enhanced cytotoxicity, showing much higher sensitivity in H1975 than A549 NSCLC cells. PIT with EITC-atezolizumab did not enhance gelonin efficacy. Confocal microscopy of H1975 cells revealed near-plasmamembrane puncta fluorescence of Alexa647-atezolizumab, with fractions clustering in CD63+ organelles peaking at 6 h. In A549 cells, AlexaFluor647-atezolizumab showed sustained accumulation in LysoTracker™ Blue+ vesicles for up to 24 h. CONCLUSION:To the best of our knowledge, this is the first documentation demonstrating that atezolizumab is transported to CD63-positive organelles, thereby enhancing our understanding of its intracellular trafficking. Our study also strengthens the concept of PIT and atezolizumab-based targeting of PD-L1+ NSCLCs.

Neural stem cells (NSCs) derived from human induced pluripotent stem cells were used to investigate effects of exposure to the food contaminant acrylamide (AA) and its main metabolite glycidamide (GA) on key neuro-developmental processes. Diet is an important source of human AA exposure for pregnant women, and AA is known to pass the placenta and the newborn may also be exposed through breast feeding after birth. The NSCs were exposed to AA and GA (1 x10(-8) - 3 x10(-3) M) under 7 days of proliferation and up to 28 days of differentiation towards a mixed culture of neurons and astrocytes. Effects on cell viability was measured using Alamar B1ueTM cell viability assay, alterations in gene expression were assessed using real time PCR and RNA sequencing, and protein levels were quantified using immunocytochemistry and high content imaging. Effects of AA and GA on neumdevelopmental processes were evaluated using endpoints linked to common key events identified in the existing developmental neumtoxicity adverse outcome pathways (AOPs). Our results suggest that AA and GA at low concentrations (1 x10(-7) - 1 x10(-8) M) increased cell viability and markers of proliferation both in proliferating NSCs (7 days) and in maturing neurons after 14-28 days of differentiation. IC50 for cell death of AA and GA was 5.2 x 10(-3) M and 5.8 x 10(-4) M, respectively, showing about ten times higher potency for GA. Increased expression of brain derived neurotrophic factor (BDNF) concomitant with decreased synaptogenesis were observed for GA exposure (10(-7) M) only at later differentiation stages, and an increased number of astrocytes (up to 3-fold) at 14 and 21 days of differentiation. Also, AA exposure gave tendency towards decreased differentiation (increased percent Nestin positive cells). After 28 days, neurite branch points and number of neurites per neuron measured by microtubule-associated protein 2 (Map2) staining decreased, while the same neurite features measured by beta III-Tubulin increased, indicating perturbation of neuronal differentiation and maturation.

Clinical parameters have been extensively studied in factor (F) VII deficiency, but the knowledge of molecular mechanisms of this disease is scarce. We report on three probands with intracranial bleeds at an early age, one of which had concomitant high titer of FVII inhibitor. The aim of the present study was to identify the causative mutations and to elucidate the underlying molecular mechanisms. All nine F7 exons were sequenced in the probands and the closest family members. A homozygous deletion in exon 1, leading to a frame shift and generation of a premature stop codon (p.C10Pfs*16), was found in proband 1. Probands 2 and 3 (siblings) were homozygous for a missense mutation in exon 8, resulting in a glycine (G) to arginine (R) substitution at amino acid 240 (p.G240R). All probands had severely reduced FVII activity (FVII:C<1IU/dL). Treatment consisted of recombinant FVIIa and/or plasma concentrate, and proband 1 developed a FVII inhibitor shortly after initiation of treatment. The FVII variants were overexpressed in mammalian cell lines. No FVII protein was produced in cells expressing the p.C10Pfs*16 variant, and the inhibitor development in proband 1 was likely linked to the complete absence of circulating FVII. Structural analysis suggested that the G to R substitution in FVII found in probands 2 and 3 would destabilize the protein structure, and cell studies demonstrated a defective intracellular transport and increased endoplasmic reticulum stress. The molecular mechanism underlying the p.G240R variant could be reduced secretion caused by protein destabilization and misfolding.

(1) Background: Congenital factor (F) VII deficiency is caused by mutations in the F7 gene. Patients with modest differences in FVII levels may display large differences in clinical severity. The variant p.A354V-p.P464Hfs is associated with reduced FVII antigen and activity. The aim of the study was to investigate the clinical manifestation of this variant and the underlying molecular mechanisms. (2) Methods: Analyses were conducted in 37 homozygous patients. The recombinant variant was produced in mammalian cells. (3) Results: We report a large variation in clinical phenotypes, which points out genetic and acquired components beyond F7 mutations as a source of variability. In contrast, patients displayed similarly reduced FVII plasma levels with antigen higher than its activity. Comparative analysis of the recombinant variant and of plasma samples from a subset of patients indicated the presence of an elongated variant with indistinguishable migration. Treatment of cells with the chemical chaperone 4-phenylbutyrate (4-PBA) improved the intracellular trafficking of the variant and increased its secretion to the conditioned medium up to 2-fold. However, the effect of 4-PBA on biological activity was marginal. (4) Conclusions: Chemical chaperones can be used as biochemical tools to study the intracellular fate of a trafficking-defective FVII variant.

In the present work, we study the photodynamic action of cercosporin (cerco), a naturally occurring photosensitizer, on human cancer multicellular spheroids. U87 spheroids exhibit double the uptake of cerco than T47D and T98G spheroids as shown by flow cytometry on the single cell level. Moreover, cerco is efficiently internalized by cells throughout the spheroid as shown by confocal microscopy, for all three cell lines. Despite their higher cerco uptake, U87 spheroids show the least vulnerability to cerco-PDT, in contrast to the other two cell lines (T47D and T98G). While 300 mu m diameter spheroids consistently shrink and become necrotic after cerco PDT, bigger spheroids (>500 mu m) start to regrow following blue-light PDT and exhibit high viability. Cerco-PDT was found to be effective on bigger spheroids reaching 1mm in diameter especially under longer exposure to yellow light (similar to 590 nm). In terms of metabolism, T47D and T98G undergo a complete bioenergetic collapse (respiration and glycolysis) as a result of cerco-PDT. U87 spheroids also experienced a respiratory collapse following cerco-PDT, but retained half their glycolytic activity.

BackgroundPhotodynamic therapy (PDT) is a minimally invasive, clinically approved therapy with numerous advantages over other mainstream cancer therapies. 5-aminolevulinic acid (5-ALA)-PDT is of particular interest, as it uses the photosensitiser PpIX, naturally produced in the heme pathway, following 5-ALA administration. Even though 5-ALA-PDT shows high specificity to cancers, differences in treatment outcomes call for predictive biomarkers to better stratify patients and to also diversify 5-ALA-PDT based on each cancer's phenotypic and genotypic individualities. AimsThe present study seeks to highlight key biomarkers that may predict treatment outcome and simultaneously be exploited to overcome cancer-specific resistances to 5-ALA-PDT. Methods and ResultsWe submitted two glioblastoma (T98G and U87) and three breast cancer (MCF7, MDA-MB-231, and T47D) cell lines to 5-ALA-PDT. Glioblastoma cells were the most resilient to 5-ALA-PDT, while intracellular production of 5-ALA-derived protoporphyrin IX (PpIX) could not account for the recorded PDT responses.We identified the levels of expression of ABCG2 transporters, ferrochelatase (FECH), and heme oxygenase (HO-1) as predictive biomarkers for 5-ALA-PDT. GPX4 and GSTP1 expression vs intracellular glutathione (GSH) levels also showed potential as PDT biomarkers.For T98G cells, inhibition of ABCG2, FECH, HO-1, and/or intracellular GSH depletion led to profound PDT enhancement. Inhibition of ABCG2 in U87 cells was the only synergistic adjuvant to 5-ALA-PDT, rendering the otherwise resistant cell line fully responsive to 5-ALA-PDT.ABCG2 or FECH inhibition significantly enhanced 5-ALA-PDT-induced MCF7 cytotoxicity, while for MDA-MB-231, ABCG2 inhibition and intracellular GSH depletion conferred profound synergies. FECH inhibition was the only synergism to ALA-PDT for the most susceptible among the cell lines, T47D cells. ConclusionThis study demonstrates the heterogeneity in the cellular response to 5-ALA-PDT and identifies biomarkers that may be used to predict treatment outcome. The study also provides preliminary findings on the potential of inhibiting specific molecular targets to overcome inherent resistances to 5-ALA-PDT.

Introduction To date studies on factor (F)VII and other hepatic vitamin K-dependent coagulation factors have relied on cell lines overexpressing these human genes. Even though these models have provided insight into the biology of these factors, they do not fully illustrate the in vivo situation. Thus, a relevant physiological model that mimics the in vivo processing of FVII in liver cells with potential for therapeutic use is needed. Methods Human induced pluripotent stem cells (hiPSCs) were differentiated into hepatocyte-like cells (iHLCs) using a non-transcription factor based, small molecule approach. Cells were grown in medium with vitamin K to ensure a correct gamma-carboxylation. Cellular FVII mRNA and protein were determined by RT-qPCR and proteomic and Western blot (WB), respectively. Secreted FVII antigen was measured by ELISA and WB and FVII activity was assessed by chromogenic assay and thrombin generation assay (TGA). Post-translational modifications of FVII protein (glycosylation) were studied using digestion with N-glycosylase F (PNGase F) and neuraminidase. Confocal immunofluorescence microscopy was used to assess the cellular expression of FVII and other vitamin K- dependent coagulation factors and inhibitors. Human primary hepatocytes or human plasma pool were used as a control in the assays. Results The resulting iHLCs expressed FVII mRNA in comparable levels to primary hepatocytes and cellular FVII peptides were identified by mass spectrometry studies. iHLCs secreted FVII at levels of around 70% compared to primary hepatocytes with detectable activity around 35% of the FVII activity level from primary hepatocytes. The TGA showed that cell medium from iHLCs when mixed with FVII deficient plasma was able to induce thrombin generation faster than the FVII depleted plasma alone (lagtime 3.2 vs 27.6 s, respectively). PNGase-F treatment showed that FVII secreted by iHLCs was N-glycosylated. Intracellular FVII was detected by WB as a band of approximately 63 kDa, slightly larger than FVII from plasma pool but similar to FVII from primary hepatocytes. Moreover, additional coagulation factors and inhibitors such as FII, FX, protein C and antithrombin were detected both at the mRNA and protein levels in the cells. Conclusions Stem cell-derived iHLCs produce and secrete FVII at physiologically relevant levels. The resulting FVII showed similar post-translational modifications to plasma FVII although some differences in proteolysis could be inferred. This iHLCs-derived FVII is able to initiate the extrinsic coagulation pathway. Our data support that these iHLCs can serve as a highly relevant model to study FVII and other vitamin K-dependent coagulation factors in vitro and constitute an important step towards the development of novel cell-based therapies for both FVII and other vitamin K-dependent coagulation factor deficiencies. Disclosures No relevant conflicts of interest to declare.

The ESCRT-III membrane fission machinery maintains the integrity of the nuclear envelope. Although primary nuclei resealing takes minutes, micronuclear envelope ruptures seem to be irreversible. Instead, micronuclear ruptures result in catastrophic membrane collapse and are associated with chromosome fragmentation and chromothripsis, complex chromosome rearrangements thought to be a major driving force in cancer development. Here we use a combination of live microscopy and electron tomography, as well as computer simulations, to uncover the mechanism underlying micronuclear collapse. We show that, due to their small size, micronuclei inherently lack the capacity of primary nuclei to restrict the accumulation of CHMP7–LEMD2, a compartmentalization sensor that detects loss of nuclear integrity. This causes unrestrained ESCRT-III accumulation, which drives extensive membrane deformation, DNA damage and chromosome fragmentation. Thus, the nuclear-integrity surveillance machinery is a double-edged sword, as its sensitivity ensures rapid repair at primary nuclei while causing unrestrained activity at ruptured micronuclei, with catastrophic consequences for genome stability. Vietri et al. show that the micronucleus fails to restrain ESCRT-III spreading due to its small size, resulting in aberrant accumulation of ESCRT-III to drive micronuclear collapse and DNA fragmentation.

Triple-negative breast cancer (TNBC) and malignant melanoma are highly aggressive cancers that widely express the cell surface chondroitin sulfate proteoglycan 4 (CSPG4/NG2). CSPG4 plays an important role in tumor cell growth and survival and promotes chemo- and radiotherapy resistance, suggesting that CSPG4 is an attractive target in cancer therapy. In the present work, we applied the drug delivery technology photochemical internalization (PCI) in combination with the novel CSPG4-targeting immunotoxin 225.28-saporin as an efficient and specific strategy to kill aggressive TNBC and amelanotic melanoma cells. Light-activation of the clinically relevant photosensitizer TPCS2a (fimaporfin) and 225.28-saporin was found to act in a synergistic manner, and was superior to both PCI of saporin and PCI-no-drug (TPCS2a + light only) in three TNBC cell lines (MDA-MB-231, MDA-MB-435 and SUM149) and two BRAFV600E mutated malignant melanoma cell lines (Melmet 1 and Melmet 5). The cytotoxic effect was highly dependent on the light dose and expression of CSPG4 since no enhanced cytotoxicity of PCI of 225.28-saporin compared to PCI of saporin was observed in the CSPG4-negative MCF-7 cells. The PCI of a smaller, and clinically relevant CSPG4-targeting toxin (scFvMEL-rGel) validated the CSPG4-targeting concept in vitro and induced a strong inhibition of tumor growth in the amelanotic melanoma xenograft A-375 model. In conclusion, the combination of the drug delivery technology PCI and CSPG4-targeting immunotoxins is an efficient, specific and light-controlled strategy for the elimination of aggressive cells of TNBC and malignant melanoma origin. This study lays the foundation for further preclinical evaluation of PCI in combination with CSPG4-targeting.

Inhibitory signaling during natural killer (NK) cell education translates into increased responsiveness to activation; however, the intracellular mechanism for functional tuning by inhibitory receptors remains unclear. Secretory lysosomes are part of the acidic lysosomal compartment that mediates intracellular signalling in several cell types. Here we show that educated NK cells expressing self-MHC specific inhibitory killer cell immunoglobulin-like receptors (KIR) accumulate granzyme B in dense-core secretory lysosomes that converge close to the centrosome. This discrete morphological phenotype is independent of transcriptional programs that regulate effector function, metabolism and lysosomal biogenesis. Meanwhile, interference of signaling from acidic Ca2+ stores in primary NK cells reduces target-specific Ca2+-flux, degranulation and cytokine production. Furthermore, inhibition of PI(3,5)P2 synthesis, or genetic silencing of the PI(3,5)P2-regulated lysosomal Ca2+-channel TRPML1, leads to increased granzyme B and enhanced functional potential, thereby mimicking the educated state. These results indicate an intrinsic role for lysosomal remodeling in NK cell education.

Congenital coagulation factor (F) VII deficiency is a rare bleeding disorder caused by mutations in the F7 gene. The missense factor FVII variant p.Q160R is the disease-causing mutation in all Norwegian FVII deficient patients and results in reduced biological activity and antigen levels of FVII in patient plasma. Previous in vitro studies on this variant demonstrated impaired intracellular trafficking and reduced secretion, possibly due to protein misfolding. The aim of the study was therefore to assess the impact of chemical chaperones on cellular processing and secretion of this variant using a cell model based on overexpression of the recombinant protein. Through screening of compounds, we identified 4-phenylbutyrate (4-PBA) to increase the secretion of recombinant (r) FVII-160R by ~ 2.5-fold. Additionally, treatment with 4-PBA resulted in a modest increase in specific biological activity. Intracellular localization studies revealed that upon treatment with 4-PBA, rFVII-160R was secreted through Golgi and Golgi reassembly-stacking protein (GRASP)-structures. The present study demonstrates that the chemical chaperone 4-PBA, restores intracellular trafficking and increases the secretion of a missense FVII variant with functional properties in the extrinsic coagulation pathway.

Inhibitory signaling during natural killer (NK) cell education translates into increased responsiveness to activation; however the intracellular basis for functional tuning by inhibitory receptors remains unclear. We found that NK cells expressing self-MHC specific inhibitory killer cell immunoglobulin-like receptors (KIR) show a greater accumulation of dense-core secretory granules, converged closer to the centrosome in resting NK cells, which are released upon recognition of target cells. This discrete morphological phenotype persists in self-KIR+ NK cells independently of transcriptional programs that regulate metabolism and granule biogenesis and indicates an intrinsic role for lysosomal homeostasis in NK cell education. Upon activation, interference of signaling from acidic Ca2+ stores reduced both target-specific Ca2+-flux, degranulation and cytokine production. Furthermore, inhibition of PI(3,5)P2 synthesis or genetic silencing of the PI(3,5)P2-regulated lysosomal Ca2+-channel TRPML1 in primary NK cells led to an increase in granular load and enhanced functional potential. These results suggest a model where continuous unopposed signaling through activating receptors render NK cells hypofunctional through TRPML1-mediated modulation of acidic Ca2+ stores.

The F7 gene mutations p.Q160R and p.A354V-p.P464Hfs are associated with very low factor VII (FVII) activity and antigen levels and a bleeding phenotype. We have previously demonstrated reduced secretion of the recombinant (r) variants rFVII-160R and rFVII-354V-464Hfs in vitro, possibly caused by a misfolding mechanism resulting in intracellular retention and increased endoplasmic reticulum (ER) stress. Chemical chaperones are compounds that can enable proteins to recover from a misfolded state and rescue intracellular processing. The aim of the study was to determine whether chemical chaperones could increase the secretion and enhance the biological activity of the above mentioned FVII mutants.

Interactions between stromal cells and tumor cells pay a major role in cancer growth and progression. This is reflected in the composition of anticancer drugs which includes compounds directed towards the immune system and tumor-vasculature in addition to drugs aimed at the cancer cells themselves. Drug-based treatment regimens are currently designed to include compounds targeting the tumor stroma in addition to the cancer cells. Treatment limiting adverse effects remains, however, one of the major challenges for drug-based therapy and novel tolerable treatment modalities with diverse high efficacy on both tumor cells and stroma is therefore of high interest. It was hypothesized that the vascular targeted fusion toxin VEGF(121)/rGel in combination with the intracellular drug delivery technology photochemical internalization (PCI) stimulate direct cancer parenchymal cell death in addition to inhibition of tumor perfusion, and that an immune mediated response is relevant for treatment outcome. The aim of the present study was therefore to elucidate the anticancer mechanisms of VEGF(121)/rGel-PCI. In contrast to VEGF(121)/rGel monotherapy, VEGF(121)/rGel-PCI was found to mediate its effect through VEGFR1 and VEGFR2, and a targeted treatment effect was shown on two VEGFR1 expressing cancer cell lines. A cancer parenchymal treatment effect was further indicated on H&E stains of CT26-CL25 and 4 T1 tumors. VEGF(121)/rGel-PCI was shown, by dynamic contrast enhanced MRI, to induce a sustained inhibition of tumor perfusion in both tumor models. A 50% complete remission (CR) of CT26. CL25 colon carcinoma allografts was found in immunocompetent mice while no CR was detected in CT26. CL25 bearing athymic mice. In conclusion, the present report indicate VEGF(121)/rGel -PCI as a treatment modality with multimodal tumor targeted efficacy that should be further developed towards clinical utilization.

Congenital factor (F) VII deficiency is a bleeding disorder caused by a heterogeneous pattern of mutations in the F7 gene. Protein misfolding due to mutations is a strong candidate mechanism to produce the highly represented type I FVII deficiency forms, characterized by a concomitant deficiency of FVII antigen and activity. Misfolded proteins can accumulate within the endoplasmic reticulum (ER) causing ER stress with subsequent activation of the unfolded protein response (UPR). So far, there are limited data on this important issue in FVII deficiency. In this study, we chose as candidate FVII model mutations, the p.Q160R, p.I289del and p.A354V-p.P464Hfs, which are all associated with severe to moderate type I FVII deficiency. In vitro expression of the recombinant (r) mutants rFVII-160R, rFVII-289del or rFVII-354V-464Hfs, which are characterized by either amino acid substitution, deletion, or by an extended carboxyl terminus, demonstrated inefficient secretion of the mutant proteins, probably caused by intracellular retention and association with ER chaperones. Both ER stress and UPR were activated following expression of all FVII mutants, with the highest response for rFVII-289del and rFVII-354V-464Hfs. These data unravel new knowledge on pathogenic mechanisms leading to FVII deficiency, and support the investigation of pharmaceutical modulators of ER stress and UPR as therapeutic agents.

Inhibitory signaling during natural killer (NK) cell education translates into increased responsiveness to activation; however the intracellular mechanism for functional tuning by inhibitory receptors remains unclear. We found that educated NK cells expressing self-MHC specific inhibitory killer cell immunoglobulin-like receptors (KIR) show accumulation of granzyme B, localized in dense-core secretory lysosomes, converged close to the centrosome. This discrete morphological phenotype persists in self-KIR + NK cells independently of transcriptional programs that regulate effector function, metabolism and lysosomal biogenesis. The granzymeB dense, large secretory lysosomes in self-KIR + NK cells were efficiently released upon target cell recognition, contributing to their enhanced cytotoxic capacity. Secretory lysosomes are part of the acidic lysosomal compartment, which has been shown to channel calcium and mediate intracellular signalling in several cell types. Interference of signaling from acidic Ca 2+ stores in primary NK cells reduced both target-specific Ca 2+ -flux, degranulation and cytokine production. Furthermore, inhibition of PI(3,5)P 2 synthesis or genetic silencing of the PI(3,5)P 2 -regulated lysosomal Ca 2+ -channel TRPML1 led to increased levels of granzyme B and enhanced functional potential. These results indicate an intrinsic role for lysosomal homeostasis in NK cell education.

Activated factor (F) VII is a vitamin K-dependent glycoprotein that initiates blood coagulation upon interaction with tissue factor. FVII deficiency is the most common of the rare congenital bleeding disorders. While the mutational pattern has been extensively characterized, the pathogenic molecular mechanisms of mutations, particularly at the intracellular level, have been poorly defined. Here, we aimed at elucidating the mechanisms underlying altered FVII biosynthesis in the presence of three mutation types in the catalytic domain: a missense change, a microdeletion and a frameshift/elongation, associated with severe or moderate to severe phenotypes. Using CHO-K1 cells transiently transfected with expression vectors containing the wild-type FVII cDNA (FVIIwt) or harboring the p.I289del, p.G420V or p.A354V-p.P464Hfs mutations, we found that the secretion of the FVII mutants was severely decreased compared to FVIIwt. The synthesis rate of the mutants was slower than the FVIIwt and delayed, and no degradation of the FVII mutants by proteasomes, lysosomes or cysteine proteases was observed. Confocal immunofluorescence microscopy studies showed that FVII variants were localized into the endoplasmic reticulum (ER) but were not detectable within the Golgi apparatus. These findings suggested that a common pathogenic mechanism, possibly a defective folding of the mutant proteins, was triggered by the FVII mutations. The misfolded state led to impaired trafficking of these proteins causing ER retention, which would explain the low to very low FVII plasma levels observed in patients carrying these mutations.

Oksvold MP, Skarpen E, Lindeman B, Roos N, Huitfeldt HS. Immunocytochemical localization of Shc and activated EGF receptor in early endosomes after EGF stimulation of HeLa cells. J Histochem Cytochem. 2000;48:21-33. (Original DOI: 10.1177/002215540004800103)