PD-L1 overexpression induces STAT signaling and promotes the secretion of pro-angiogenic cytokines in non-small cell lung cancer (NSCLC).

BACKGROUND: Monoclonal antibodies (ICI) targeting the immune checkpoint PD-1/PD-L1 alone or in combination with chemotherapy have demonstrated relevant benefits and established new standards of care in first-line treatment for advanced non-oncogene addicted non-small cell lung cancer (NSCLC). However, a relevant percentage of NSCLC patients, even with high PD-L1 expression, did not respond to ICI, highlighting the presence of intracellular resistance mechanisms that could be dependent on high PD-L1 levels. The intracellular signaling induced by PD-L1 in tumor cells and their correlation with angiogenic signaling pathways are not yet fully elucidated. METHODS: The intrinsic role of PD-L1 was initially checked in two PD-L1 overexpressing NSCLC cells by transcriptome profile and kinase array. The correlation of PD-L1 with VEGF, PECAM-1, and angiogenesis was evaluated in a cohort of advanced NSCLC patients. The secreted cytokines involved in tumor angiogenesis were assessed by Luminex assay and their effect on Huvec migration by a non-contact co-culture system. RESULTS: PD-L1 overexpressing cells modulated pathways involved in tumor inflammation and JAK-STAT signaling. In NSCLC patients, PD-L1 expression was correlated with high tumor intra-vasculature. When challenged with PBMC, PD-L1 overexpressing cells produced higher levels of pro-angiogenic factors compared to parental cells, as a consequence of STAT signaling activation. This increased production of cytokines involved in tumor angiogenesis largely stimulated Huvec migration. Finally, the addition of the anti-antiangiogenic agent nintedanib significantly reduced the spread of Huvec cells when exposed to high levels of pro-angiogenic factors. CONCLUSIONS: In this study, we reported that high PD-L1 modulates STAT signaling in the presence of PBMC and induces pro-angiogenic factor secretion. This could enforce the role of PD-L1 as a crucial regulator of the tumor microenvironment stimulating tumor progression, both as an inhibitor of T-cell activity and as a promoter of tumor angiogenesis.

Soluble PD-L1 and Circulating CD8+PD-1+ and NK Cells Enclose a Prognostic and Predictive Immune Effector Score in Immunotherapy Treated NSCLC patients.

INTRODUCTION: Upfront criteria to foresee immune checkpoint inhibitors (ICIs) efficacy are far from being identified. Thus, we integrated blood descriptors of pro-inflammatory/immunosuppressive or effective anti-tumor response to non-invasively define predictive immune profiles in ICI-treated advanced non-small cell lung cancer (NSCLC). METHODS: Peripheral blood (PB) was prospectively collected at baseline from 109 consecutive NSCLC patients undergoing ICIs as first or more line treatment. Soluble PD-L1 (sPD-L1) (immunoassay), CD8+PD-1+ and NK (FACS) cells were assessed and interlaced to generate an Immune effector Score (IeffS). Lung Immune Prognostic Index (LIPI) was computed by LDH levels and derived Neutrophil-to-Lymphocyte Ratio (dNLR). All these parameters were correlated with survival outcome and treatment response. RESULTS: High sPD-L1 and low CD8+PD-1+ and NK number had negative impact on PFS (P < 0.001), OS (P < 0.01) and ICI-response (P < 0.05). Thus, sPD-L1high, CD8+PD-1+low and NKlow were considered as risk factors encompassing IeffS, whose prognostic power outperformed that of individual features and slightly exceeded that of LIPI. Accordingly, the absence of these risk factors portrayed a favorable IeffS characterizing patients with significantly (P < 0.001) prolonged PFS (median NR vs 2.3 months) and OS (median NR vs 4.1) and greater benefit from ICIs (P < 0.01). We then combined each risk parameter composing IeffS and LIPI (LDHhigh, dNLRhigh), thus defining three distinct prognostic classes. A remarkable impact of IeffS-LIPI integration was documented on survival outcome (PFS, HR = 4.61; 95%CI = 2.32-9.18; P < 0.001; OS, HR=4.03; 95%CI=1.91-8.67; P < 0.001) and ICI-response (AUC=0.90, 95%CI=0.81-0.97, P < 0.001). CONCLUSION: Composite risk models based on blood parameters featuring the tumor-host interaction might provide accurate prognostic scores able to predict ICI benefit in NSCLC patients.

Galectin-1 suppression delineates a new strategy to inhibit myeloma-induced angiogenesis and tumoral growth in vivo.

Galectin-1 (Gal-1) is involved in tumoral angiogenesis, hypoxia and metastases. Actually the Gal-1 expression profile in multiple myeloma (MM) patients and its pathophysiological role in MM-induced angiogenesis and tumoral growth are unknown. In this study, we found that Gal-1 expression by MM cells was upregulated in hypoxic conditions and that stable knockdown of hypoxia inducible factor-1α significantly downregulated its expression. Therefore, we performed Gal-1 inhibition using lentivirus transfection of shRNA anti-Gal-1 in human myeloma cell lines (HMCLs), and showed that its suppression modified transcriptional profiles in both hypoxic and normoxic conditions. Interestingly, Gal-1 inhibition in MM cells downregulated proangiogenic genes, including MMP9 and CCL2, and upregulated the antiangiogenic ones SEMA3A and CXCL10. Consistently, Gal-1 suppression in MM cells significantly decreased their proangiogenic properties in vitro. This was confirmed in vivo, in two different mouse models injected with HMCLs transfected with anti-Gal-1 shRNA or the control vector. Gal-1 suppression in both models significantly reduced tumor burden and microvascular density as compared with the control mice. Moreover, Gal-1 suppression induced smaller lytic lesions on X-ray in the intratibial model. Overall, our data indicate that Gal-1 is a new potential therapeutic target in MM blocking angiogenesis.

Combination of letrozole, metronomic cyclophosphamide and sorafenib is well-tolerated and shows activity in patients with primary breast cancer.

PURPOSE: To assess whether the combination of letrozole, metronomic cyclophosphamide and sorafenib (LCS) is well tolerated and shows activity in primary breast cancer (BC). METHODS: Thirteen oestrogen receptor-positive, postmenopausal, T2-4, N0-1 BC patients received the LCS combination for 6 months. In these patients we examined the pharmacokinetics of sorafenib and cyclophosphamide, toxicity of the regimen, the clinical response to therapy and changes in the levels of biologically relevant biomarkers. RESULTS: Adequate plasma concentrations of sorafenib were achieved in patients when it was dosed in combination with L+C. The mean plasma concentrations of C were consistently lower following administration of LCS, compared with administration of L+C only. The most common drug-related grade 3/4 adverse events were skin rash (69.3%), hand-foot skin reaction (69.3%) and diarrhoea (46.1%). According to RECIST Criteria, a clinical complete response was observed in 6 of 13 patients. A significant reduction in tumour size, evaluated with MRI, was also observed between baseline and 14 days of treatment in all 13 patients (P=0.005). A significant reduction in SUV uptake, measured by (18)FDG-PET/CT, was observed in all patients between baseline and 30 days of treatment (P=0.015) and between baseline and definitive surgery (P=0.0002). Using modified CT Criteria, a response was demonstrated in 8 out of 10 evaluable patients at 30 days and in 11 out of 13 evaluable patients at the definitive surgery. A significant reduction in Ki67 expression was observed in all patients at day 14 compared with baseline (P<0.00001) and in 9 out of 13 patients at the definitive surgery compared with baseline (P<0.03). There was also a significant suppression of CD31 and VEGF-A expression in response to treatment (P=0.01 and P=0.007, respectively). CONCLUSIONS: The LCS combination is feasible and tolerable. The tumour response and target biomarker modulation indicate that the combination is clinically and biologically active.

Epidermal growth factor receptor irreversible inhibitors: chemical exploration of the cysteine-trap portion.

Covalent EGFR irreversible inhibitors showed promising potential for the treatment of gefitinib-resistant tumors and for imaging purposes. They contain a cysteine-reactive portion forming a covalent bond with the protein. Irreversible kinase inhibitors have been advanced to clinical studies, mostly characterized by an acrylamide or butynamide warhead. However, the clinical usefulness of these compounds has been hampered by resistances, toxicity and pharmacokinetic problems. Investigation on the structure-activity and structure-reactivity relationships may provide useful information for compounds with improved selectivity and pharmacokinetic properties. This review focuses on the exploration of the cysteine-trap portions able to irreversibly inhibit EGFR and other erbB receptors.

Effect of inducible FHIT and p53 expression in the Calu-1 lung cancer cell line.

Loss of FHIT expression and p53 mutations are critical events in the early stages of lung carcinogenesis. The restoration of Fhit function in FHIT-negative cancer cells has been reported to cause tumour suppression by inhibition of cell proliferation and/or activation of apoptotic pathways. However, the studies designed to elucidate the biological role of Fhit and its potential interaction with p53 have produced conflicting results. We investigated here the effects of the simultaneous restoration of FHIT and p53 in Calu-1 cells by using a hormone-inducible gene expression system. We demonstrate that the restoration of FHIT expression reinforces the anti-proliferative effect associated with the simultaneous replacement of p53. Indeed, a more pronounced inhibition of cell proliferation associated with an earlier and higher induction of p21(waf1) mRNA and protein expression was observed in Fhit/p53-expressing cells compared with cells expressing p53 alone. This effect was not due to Fhit-mediated up-regulation of p53 expression; in fact p53 protein was expressed at the same level in both FHIT-positive and FHIT-negative cell clones. Consistent with this result, Fhit did not affect the expression of MDM2, a protein known to interact directly with p53 and target p53 for proteolytic degradation, thus down-regulating its activity.

The effect of heat shock on amino acid transport and cell volume in 3T3 cells.

In 3T3 cells temperatures higher than physiological stimulated amino acid transport activity in a dose-dependent manner up to 44 degrees C. However, the temperature increase did not induce widespread transport increase of all other nutrients tested. The activities of both amino acid transport systems A and ASC were enhanced within a few minutes following cell exposure to increased temperature. The maintenance of this effect required continuous exposure of the cells to hyperthermia. Kinetic analysis indicated that the stimulation of the activity of transport System A occurred through a mechanism affecting Vmax rather than Km. The continuous presence of cycloheximide did not prevent the transport changes induced by hyperthermia. These results suggest that the increased amino acid uptake reflects an activation or relocation of existing amino acid transport proteins. During the hyperthermic treatment, the content of ninhydrin-positive substances (NPS), mostly amino acids, increased within the cells and the accumulation of these compatible osmolytes was parallelled by an increase in cell volume. The withdrawal of amino acids from the culture medium immediately before and during the shock phase counteracted the increase and reduced the NPS content but did not prevent the increase in amino acid transport, the cell swelling and the induction of the heat shock response.

Heat-induced proteasomic degradation of HSF1 in serum-starved human fibroblasts aging in vitro.

The exposure of human fibroblasts (HF) aging in vitro to heat shock resulted in an attenuated expression of the heat shock-inducible HSP70. When late passage cells were cultured in the continuous presence of serum, we observed a reduced accumulation of the cytoplasmic polyadenylated HSP70 mRNA. The levels of HSF1 activation and nuclear HSP70 mRNA were comparable to those of early passage cells (M. A. Bonelli et al., Exp. Cell Res. 252, 20-32, 1999). When late passage cells were serum-starved overnight, we observed a reduced activation of HSF1 and a decreased level of HSP70 mRNA during heat shock. However, at 37 degrees C the levels of HSF1 differed little between late passage HF and early passage cells, irrespective of the presence of serum. Interestingly, during heat shock a marked decrease in the level and, consequently, in the binding activity of HSF1 was noted only in serum-starved, late passage HF. The decrease in the level of HSF1 was counteracted by back addition of serum to the cells during heat shock. Addition of the specific proteasome inhibitor MG132 blocked a decrease in HSF1 during heat shock, maintaining levels observed in late passage cells and HSF1 activity comparable to that of early passage HF. The recovery of the level and activity of HSF1 observed in late passage HF incubated in the presence of MG132 suggests that heat shock unmasks a latent proteasome activity responsible for HSF1 degradation.

Osmotic regulation of ATA2 mRNA expression and amino acid transport System A activity.

When porcine endothelial cells were exposed to hypertonicity, both the level of ATA2 (amino acid transporter 2) mRNA and activity of amino acid transport System A increased transiently, peaking after about 6 and 9 h, respectively. Cycloheximide, like actinomycin D, prevented both responses, showing that an earlier step also involves protein synthesis. Withdrawal of hypertonicity after 6 h increased the rate of down regulation. These findings confirm that ATA2 is a major isoform of System A and show that changes in the expression of ATA2 mRNA precede both the induction and subsequent down regulation of transport activity.

Induction of BGT-1 and amino acid system A transport activities in endothelial cells exposed to hyperosmolarity.

We studied the responses to hypertonicity of cultured endothelial cells from swine pulmonary arteries. In 0.5 osmol/kgH(2)O medium, initial cell shrinkage was followed by a regulatory volume increase (RVI), complete after 1 h, concomitant with an increase in cellular K(+) content. Then the activity of amino acid transport System A increased, accompanied by an accumulation of ninhydrin-positive solutes (NPS), reaching a peak at approximately 6 h. The subsequent decline in System A activity was paralleled by an induction of the betaine-GABA transporter (BGT-1), detected as increases of BGT-1 mRNA and of transport activity, which peaked at approximately 24 h. Inhibitors of transcription or translation prevented induction of both transport activities. The increased expression of BGT-1, which involved activation of "tonicity-responsive enhancer," was inhibited by 5 mM extracellular betaine. Cellular K(+) concentration gradually declined after the accumulation of NPS and during the induction of BGT-1. This very effective adaptation to hypertonicity suggests it has a physiological role.

Attenuated expression of 70-kDa heat shock protein in WI-38 human fibroblasts during aging in vitro.

We examined the effects of cellular aging on the expression of the heat shock-inducible HSP70 gene in WI-38 diploid human fibroblasts serially passaged in vitro. The senescence of the cells was established by evaluating population doubling level, cell density at confluency, and cell morphology along with the detection of senescence-associated beta-galactosidase activity (histochemically detectable at pH 6), a reliable marker of aging in low-density cultures. A marked decrease in the synthesis and accumulation of the inducible HSP70 protein was observed in serum-fed late passage cells exposed to a severe heat shock (30 min at 45 degrees C) in comparison to early passage cells. However, the degree of HSF-DNA binding, monitored by gel retardation assay was similar in both early and late passage cells. Similarly, Northern blotting analysis indicated that comparable amounts of inducible HSP70 mRNA were present in the total RNA fraction, in the total polyadenylated RNA fraction, or in the nuclear polyadenylated RNA fraction extracted from both early and late passage cells. In contrast, much less inducible HSP70 mRNA was detected in the total cytoplasmic RNA fraction or in the polyadenylated cytoplasmic RNA fraction of late passage cells. Thus age-related differences in heat-induced HSP70 synthesis and accumulation observed in serum-fed WI-38 cells appeared to result from an impairment in the posttranscriptional processing of the HSP70 mRNA at a level following the polyadenylation step and preceding translocation from the nucleus to the cytoplasm. When HF were serum deprived for 20 h before heat shock, the induction of HSP70 mRNA was less than 30% reduced in early passage cells in comparison to serum-fed cells; however, the level of HSP70 mRNA was markedly (over 80%) decreased in serum-deprived late passage cells. This result indicated that the presence of serum has a strong influence on heat shock-induced HSP70 gene expression in human fibroblasts aging in vitro.

Induction of betaine-gamma-aminobutyric acid transport activity in porcine chondrocytes exposed to hypertonicity.

1. We measured the rates of uptake of selected amino acids and betaine by primary cultures of chondrocytes from porcine articular cartilage after the cells had been incubated in 'isotonic' (0.3 osmol l-1) or hypertonic (0.5 osmol l-1) media. 2. Na+-dependent uptake of methylaminoisobutyric acid increased rapidly when the cells were exposed to hypertonic conditions, reached a peak after 6-9 h, and then gradually decreased so that after 24 h it was only slightly above the control value. Conversely, (Na+ + Cl-)-dependent influx of gamma-aminobutyric acid (GABA) remained low for the first 9 h of hypertonic incubation, but then increased markedly to reach a plateau value after 24-30 h. Betaine influx also increased in cells incubated in hypertonic medium, being mainly Na+ dependent after 6 h, but (Na+ + Cl-)-dependent after 24 h. 3. This pattern indicates that exposure of the chondrocytes to hypertonicity induces first amino acid transport system A and then, as this decreases again, betaine-GABA transport activity. 4. Induction of betaine-GABA transport activity did not require continuous exposure of chondrocytes to hypertonicity; but the magnitude of the increase measured at the end of a 24 h incubation period was proportional to the length of time the cells had been exposed to hypertonicity during the 24 h. 5. Isolation and culture of chondrocytes in 0.4 osmol l-1 medium, instead of 0.3 osmol l-1, significantly increased their betaine-GABA transport activity, but not their system A activity. 6. Induction of betaine-GABA transport activity was prevented by addition of either actinomycin D or cycloheximide to the medium, but no mRNA for the betaine-GABA transporter known as BGT-1 was detected by Northern blot analysis of extracts of chondrocytes.

Expression of human CD44v6 in non-small-cell lung cancer.

INTRODUCTION: The CD44 is a membrane glycoprotein that functions as lymph node homing receptor in lymphocyte activation and is involved in homo- and heterotypic cell adhesion. In several tumor cell lines the expression of splice variants (CD44v6 and CD44v7) are correlated with the metastatic potential and confer an advantage in the early steps of the metastatic cascade. In our study we examined 35 cases of non-small-cell lung cancers (NSCLC) in order to detect the presence of CD44v6 and to compare its expression with the histologic type, degree of differentiation, stage of the tumor and survival of the patients. METHODS: CD44v6 expression in frozen tissue sections of 35 patients with NSCLC who underwent pneumonectomy or lobectomy was analyzed with the VFF-7 monoclonal antibody that detected the CD44v6 variant. The data on survival were analyzed by the actuarial method and compared by the log rank test. RESULTS: The expression of CD44v6 occurred in all the 20 cases of epidermoid carcinomas tested and in 2 out of the 3 cases of undifferentiated large cell carcinoma and was absent in all the 12 adenocarcinomas. No relationship was found between the presence of this marker and the grading or the stage of the pathology. The 3-year survival rate was 73% for CD44v6-positive and 65% for CD44v6-negative cancer and the comparison was not statistically significant. CONCLUSION: These results suggest that in lung cancer the expression of CD44v6 is not a useful prognostic factor.

Primer tRNA(Trp) of RSV-transformed or RAV-1-infected cells up-regulates the antiribosomal activity of gelonin.

Some ribosome-inactivating proteins (RIPs) with RNA-N-glycosidase activity on 28S rRNA require, for maximal inactivation of ribosomes, the presence of tRNA. tRNA(Trp) specifically up-regulates gelonin, the RIP from Gelonium multiflorum. The same tRNA is the primer of the reverse transcriptase of Rous sarcoma virus (RSV) and of its mutant (RAV-1) which lacks the src gene. Here we demonstrate that gelonin is more active in inhibiting endogenous protein synthesis by lysates of RSV-transformed or RAV-1-infected cells and that such increase in activity correlates with the increased amount of primer tRNA(Trp) in the cells.

An original application of plasma expanders: heart-lung preservation.

The lack of an ideal heart-lung preservation solution is one of the principal factor that limits the wide spread of transplantation. The aim of this work was to investigate the efficacy of Haemaccel (HM) on isolated human pulmonary artery endothelial cells comparing its effects with those of University of Wisconsin (UWS). Subcultures of HPAEC were inoculated at the density of 5,000 cells per cm2 in 9 cm2 well-plates. Cells were incubated with HM and UWS for 6 hrs at 10 degrees C. Cellular viability was analysed by the total protein content (cytotoxicity index) and by the rate of protein synthesis (metabolic index). The results showed that HM and UWS did no show a significant differences in the toxicity when compared with the control; on the contrary, HM seems to determine a less inhibitory effect on cellular metabolism permitting a more rapid cellular metabolic recovery than UWS. Thus, HM appears to be more suitable for the preservation of isolated HPAEC than UWS.

Cell susceptibility to apoptosis by glutamine deprivation and rescue: survival and apoptotic death in cultured lymphoma-leukemia cell lines.

Human leukemia/lymphoma cells maintained in culture medium without provision of fresh nutrients lose viability and die by a process resembling apoptosis within a few days. Upon incubation in an FCS-supplemented RPMI 1640 medium containing 2 mM L-glutamine CEM, Namalwa, HL-60 and U937 cells, seeded at initial densities of 0.2 to 1 x 10(6) cells/ml, ceased growing within 3-5 days and progressively entered an apoptotic pathway, as assessed by nucleosomal DNA fragmentation and morphology. Both the major energy-source nutrients in the medium, glucose and glutamine, became rapidly exhausted during the incubation. Further studies were performed using CEM cells. Incubation in glutamine-free or glucose-free medium renewed every 24 h showed that glutamine deprivation is associated with cell death by apoptosis independent of energetic failure, whereas glucose deprivation is followed by rapid loss of mitochondrial function with sharp drop of intracellular ATP and cell death by necrosis. A 12-24 h incubation in glutamine-depleted medium was required to direct the cells toward the apoptotic pathway. Growth arrest followed by apoptotic death was detected in CEM cells when medium glutamine concentration remained below 0.3-0.4 mM for at least 24 h, but a reinstatement of medium glutamine to 2 mM within this period rescued the cells from growth arrest and death.

Activation of heat-shock transcription factor 1 by hypertonic shock in 3T3 cells.

The exposure of 3T3 cells to a medium made hypertonic by the addition of NaCl induced activation of a heat-shock transcription factor (HSF). This activation, as monitored by gel-mobility-shift assays, occurred within 10 min of hypertonic shock and was dose-dependent in relation to the osmotic strength of the medium up to 0.7 osM. Competition analysis indicated that the effect of hypertonic shock on HSF binding activity was specific. The magnitude of the heat-shock element (HSE)-HSF binding induced by incubating the cells in a 0.7 osM medium was comparable in intensity and time course with that induced by a 44 degrees C heat shock. Following removal of the stressors, the decrease in HSF-HSE binding was more rapid in hypertonicity-shocked than in heat-shocked cells. Treatment of the cells with cycloheximide did not inhibit HSF-HSE binding, indicating that the activation was independent of new protein synthesis. By using a specifically directed polyclonal serum, HSF1 was identified as the transcription factor involved in the hypertonicity-induced activation. HSF was also activated when a membrane-impermeable osmolyte such as sucrose was used to increase the osmolarity of the medium. However, no HSF-HSE binding was observed after addition of glycerol (a freely membrane-permeable osmolyte) in excess. There was a temporal relationship between the hypertonicity-induced volume decrease, the increase in the intracellular K+ concentration and the induction of HSF-HSE binding. In contrast, an increase in the intracellular Na+ concentration was not required to induce HSF-HSE binding. However, unlike the heat-shock response, the activation of HSF by hypertonic shock did not lead to elongation of the RNA transcript of heat-shock protein 70.

Adaptive cellular response to osmotic stress in pig articular chondrocytes.

The authors studied the effects of a wide range of medium osmolarities (from 0.28 osM (physiological osmolarity of plasma and synovial fluid) to 0.58 osM) by altering Na+ concentration in high density cultures of pig articular chondrocytes in order to analyze the behaviour of some functional and structural parameters during cell adaptation to these imposed changes in the ionic environment. Biochemical and morphological results indicated that, even if isolated from the tissue matrix and cultured in vitro, chondrocytes maintained active osmoregulation systems which are present in living conditions. They showed a similar biochemical and morphological behavior when cultured at 0.28 osM and 0.38 osM but they were able, with regard to protein synthesis, aminoacid transport and proliferation rates, to respond quickly and to adapt to 0.48 osM medium as well. On the contrary, the treatment at the highest osmolarity (0.58 osM) early altered these biochemical parameters and was detrimental or even gave rise to lethal damage during long-term treatment. Furthermore, while chondrocytes cultured in 0.28-0.38 osM medium maintained phenotypic characteristics in culture, the higher osmolarities (0.48-0.58 osM) caused morphological changes in cell populations resulting in loss of phenotypic cell stability as demonstrated by their taking on a fibroblast-like shape as well as a lack of ability to assembly matrix proteoglycans.