Atezolizumab Retains Cellular Binding to Programmed Death Ligand 1 Following Aerosolization via Mesh Nebulizer.

BACKGROUND/AIM: Cytotoxic inhalable drugs were shown to be advantageous in treating malignancies of the respiratory tract. However, these drugs have not always presented a safe profile and were reported to induce local adverse events. Protein-based anticancer drugs, such as immune checkpoint and vascular endothelial growth factor inhibitors, do not induce tissue injury, nor do they exhibit vesicant properties upon direct contact with tissues. Protein drugs are susceptible to the heat and stress encountered during droplet generation for delivery by nebulization. The aim of this study was to investigate the capacity of atezolizumab, an antibody to programmed death ligand 1, to bind target cells after nebulization with a vibrating mesh (VM) nebulizer. MATERIALS AND METHODS: We compared Fourier-transformed infrared (FTIR) and Raman spectra of native atezolizumab (60 mg/ml) and its nebulized form following 10-min nebulization in a piezoceramic VM nebulizer. The binding of atezolizumab to DU-145 prostate cancer cells was evaluated using competitive blocking of anti-CD274 staining. RESULTS: Nebulization did not induce Raman or FTIR spectral modification nor did it affect the binding capacity of atezolizumab. Conversely, heat-inactivated atezolizumab lost its cell-binding capacity and did not reduce anti-CD274 immunostaining. Native and nebulized atezolizumab displayed identical spectra, whereas the FTIR spectra of the heat-inactivated drug was significantly altered. CONCLUSION: VM nebulization does not obliterate the functionality of the drug atezolizumab. The integrity of a nebulized form can be rapidly assessed by FTIR and Raman spectrometry.

Thymus Subset Alterations Accompanying Concomitant Tumor Immunity Mimics Phenotypic Patterns of Cytotoxic Drug Doxorubicin.

BACKGROUND/AIM: Concomitant immunity (CIM) is a phenomenon that elicits an antitumor response not sufficient enough to destroy the primary tumor but prevents a secondary implant from growing and spreading. This study aimed to develop a method of identification of serum tumoricidal factors released into circulation during CIM and to compare the CIM-related effect to the effect elicited by the cytotoxic drug doxorubicin. MATERIALS AND METHODS: SL2 tumor-bearing mice were studied at three time points - day 4, day 7, and day 11 following i.p. 5×105 cell implantation. Hematological effects and thymocyte immunophenotyping (CD4/CD8) data were compared to the effects induced by intravenous 10 mg/kg doxorubicin (DOX) administration to intact DBA 2 mice. The level of plasma colony stimulating factor-granulocyte macrophage (CSF-GM) was evaluated by ELISA. RESULTS: Identical thymus histopathology and an extent of double-positive CD4+CD8+ subset depletion was found in day 11 tumor-bearing mice (TBM-11) and in DOX-administered animals. TBM-11 exhibited a leukemoid reaction with an increase in monocyte and granulocyte counts. Conversely, DOX administration was followed by severe leukocytopenia at the 72-h time point. No increase in CSF-GM was observed in mice with or without a leukemoid reaction. CONCLUSION: The complexity of CIM can be examined by tracking alterations in the most fragile cortical CD8+CD4+ double positive population. Thymocyte apoptosis induced by DOX and TBM-11 might be associated with different mechanisms. TBM-11 did not exhibit severe myelotoxicity as DOX did. CIM-related serum factors can be assessed and screened via thymocyte subset analysis.

Do the benefits of being a smoker hint at the existence of PD-1/PD-L1 sensitizers for patients on single-agent immunotherapy?

Multiple studies demonstrate significantly better therapeutics outcomes in smokers as compared with never smokers when single-agent immunotherapy is applied. Non-smoker patients usually need a combination of chemoimmunotherapy to achieve comparable or slightly better therapeutic results. This effect is thought to be due to tobacco product-induced upregulation of PD-L1/PD-1 expression and tumor mutational burden score. Genomic transformation, however, cannot entirely explain the upregulation of PD-L1/PL-1 expression in cells following short-term exposure to cytotoxic compounds. Cytotoxic drugs, crude tobacco products, benzo(a)pyrene, nicotine, and multiple other toxic compounds were shown to exhibit rapid PD-L1/PD-1 upregulation. A significant immunomodulatory effect of nicotine via acetylcholine receptors is well documented. However, nicotine activity rapidly subsides when the drug is withdrawn. We hypothesize that smoking cessation might mitigate the benefits of monoimmunotherapy for some patients. Further studies of the nicotinic acetylcholine receptor stimulus of immunocytes are needed and might lead to characterization and clinical implementation of new immunotherapy sensitizer products.

Immunophenotype Rearrangement in Response to Tumor Excision May Be Related to the Risk of Biochemical Recurrence in Prostate Cancer Patients.

BACKGROUND: Prostate cancer (PCa) is known to exhibit a wide spectrum of aggressiveness and relatively high immunogenicity. The aim of this study was to examine the effect of tumor excision on immunophenotype rearrangements in peripheral blood and to elucidate if it is associated with biochemical recurrence (BCR) in high risk (HR) and low risk (LR) patients. METHODS: Radical prostatectomy (RP) was performed on 108 PCa stage pT2-pT3 patients. Preoperative vs. postoperative (one and three months) immunophenotype profile (T- and B-cell subsets, MDSC, NK, and T reg populations) was compared in peripheral blood of LR and HR groups. RESULTS: The BCR-free survival difference was significant between the HR and LR groups. Postoperative PSA decay rate, defined as ePSA, was significantly slower in the HR group and predicted BCR at cut-off level ePSA = -2.0% d-1 (AUC = 0.85 (95% CI, 0.78-0.90). Three months following tumor excision, the LR group exhibited a recovery of natural killer CD3 - CD16+ CD56+ cells, from 232 cells/µL to 317 cells/µL (p < 0.05), which was not detectable in the HR group. Prostatectomy also resulted in an increased CD8+ population in the LR group, mostly due to CD8+ CD69+ compartment (from 186 cells/µL before surgery to 196 cells/µL three months after, p < 001). The CD8+ CD69+ subset increase without total T cell increase was present in the HR group (p < 0.001). Tumor excision resulted in a myeloid-derived suppressor cell (MDSC) number increase from 12.4 cells/µL to 16.2 cells/µL in the HR group, and no change was detectable in LR patients (p = 0.12). An immune signature of postoperative recovery was more likely to occur in patients undergoing laparoscopic radical prostatectomy (LRP). Open RP (ORP) was associated with increased MDSC numbers (p = 0.002), whereas LRP was characterized by an immunity sparing profile, with no change in MDSC subset (p = 0.16). CONCLUSION: Tumor excision in prostate cancer patients results in two distinct patterns of immunophenotype rearrangement. The low-risk group is highly responsive, revealing postoperative restoration of T cells, NK cells, and CD8+ CD69+ numbers and the absence of suppressor MDSC increase. The high-risk group presented a limited response, accompanied by a suppressor MDSC increase and CD8+ CD69+ increase. The laparoscopic approach, unlike ORP, did not result in an MDSC increase in the postoperative period.

Comparative Evaluation of Cellular Uptake of Free and Liposomal Doxorubicin Following Short Term Exposure.

BACKGROUND/AIM: Liposomal Doxorubicin (lipDOX) and free Doxorubicin (DOX) are reported to exhibit similar antitumor efficacy. However, cellular internalization mechanisms of lipDOX are still a subject of controversy. MATERIALS AND METHODS: Intact and permeabilized cells were exposed for short time to lipDOX and free DOX and drug intracellular content was evaluated by flow cytometry. Then, the antiproliferative capacities of lipDOX and free DOX were compared by the leukocyte nadir test in mice in vivo. RESULTS: The fluorescence increase was 11.2-fold higher in intact cells and 19.7-fold higher in permeabilized cells after exposure to free DOX as compared to lipDOX. Mice injected with DOX showed pronounced antiproliferative activity with a leukocyte count decrease to 2.8±0.65 k/µl (p<0.01) - an effect significantly stronger than that in the lipDOX group. CONCLUSION: Intact and permeabilized cells internalize free DOX manifold faster than lipDOX. The LipDOX formulation does not induce a remarkable leukocyte nadir effect in vivo.

A Refinement of Clinical Tumor Marker Monitoring: Why Not Use an Inverse Value of Doubling Time?

OBJECTIVES: The aim of this study was to compare prostate-specific antigen (PSA) kinetics - half-life time (HT), doubling time (DT), and elimination rate PSA (ePSA) in prostate cancer (PCa) monitoring. Implementation of ePSA in clinical practice could help simplify patient monitoring in the remission phase. MATERIALS AND METHODS: A total of 49 PCa patients were examined by their PSA tests before prostatectomy and after 30 days, 91 days, and 24 months. Conventional PSA rate of change parameters (HT and DT) were compared to a new clinically understandable ePSA parameter. RESULTS: We observed that implementation of inverse value (ePSA) rather than HT or DT has distinct advantages: (1) values are valid when PSA is unchanged (ePSA equals zero), (2) the concept of ePSA can be easily understood, as it is a growth fraction, (3) ePSA fluctuates within a narrow range and is thus easy to interpret, and (4) there are no mathematical flaws (no positive skewing). CONCLUSION: Exploring ePSA norm as ≤0% could help spot biochemical recurrence in a timely manner. Primary health care providers tend to use an irrelevant PSA threshold, that is, 4.0 ng/mL, in postoperative follow-up. The delayed referrals of patients in remission might be reduced if ePSA testing is adopted.

Doxorubicin uptake in ascitic lymphoma model: resistance or curability is governed by tumor cell density and prolonged drug retention.

Background/Aims: Chemotherapy resistance of malignancies is a universal phenomenon which unfavorably affects therapeutic results. Genetic adaptations as well as epigenetic factors can play an important role in the development of multidrug resistance. Cytotoxic drug content in plasma of cancer patients is known to variate up to one hundred-fold regardless of the same dose injected per m2 body surface. The relationship between plasma concentrations, tissue uptake, and chemotherapy response is not completely understood. The main objective of this study was to investigate how the identical dose of Doxorubicin (Dox) can result in a different therapeutic response pattern depending on tumor size. Study Design: The study was performed on ascitic EL4 lymphoma in an exponential growth phase focusing on the rapidly changing tumor susceptibility to the Dox treatment. Well distinguishable tumor response patterns (curability, remission-relapse, resistance) were selected to unveil Dox intratumoral uptake and drug tissue persistence. Intratumoral Dox content within peritoneal cavity (PerC) in conjunction with systemic toxicity and plasma pharmacokinetics, were monitored at several time points following Dox injection in tumor bearing mice (TBM) with differing patterns of response. Results: Following intraperitoneal (i.p.) transplantation of 5x104 EL4 lymphoma cells rapid exponential proliferation with ascites volume and animal mass increase resulted in median survival of 14.5 days. The increase in tumor cell mass in PerC between day 3 and day 9 was 112.5-fold (0.2±0.03 mg vs 22.5±0.31 mg respectively). However, tumors at this time interval (day 3 to day 9 post-transplantation) were relatively small and constituted less than 0.05% of animal weight. An identical dose of Dox (15 mg/kg) injected intravenously (i.v.) on Day 3 lead to a cure whereas a TBM injected on day 9 exhibited resistance with a median survival time no different from the untreated TBM control. Injection of Dox resulted in noticeable differences of cellular uptake in PerC between all three groups of TBM ("cure", relapse", "resistance"). Larger tumors were consistently taking up less Dox 60 min after the 15 mg/kg i.v. bolus injection. Higher initial uptake resulted also in longer retention of drug in PerC cells. The area under the concentration curve in PerC cells AUC0-10d was 8.2±0.57 µg/g x h, 4.6±0.27 µg/g x h and 1.6±0.02 µg/g x h in "cure", "relapse" and "resistance" TBM respectively (p<0.05 "relapse" vs "cure" and p<0.001 "resistance" vs "cure"). No differences in plasma Dox pharmacokinetics or systemic hematological effects were observed in TBM following a single i.v. Dox push. Hematologic nadir was tested on day 2 and subsequent hematologic recovery was evaluated on day 10 following Dox administration. Hematologic recovery on day 10 coincided with complete drug efflux from PerC and rising tumor cell numbers in PerC of "relapse" TBM. Myelosuppression and hematological recovery patterns were identical in all surviving animal groups regardless of the tumor size on the day of Dox injection. Conclusions: Within a few days of exponential tumor growth, an identical dose of Dox produced dramatically different responses in the TBM with increasing resistance. Systemic toxicity and plasma pharmacokinetics were indistinguishable between all TBM groups. Initial uptake in tumor cells was found to be consistently lower in larger tumors. Drug uptake in tumor cells was regulated locally - a phenomenon known as inoculum effect in vitro. The duration of drug retention in cells was directly related to initial cellular uptake. The magnitude of Dox cellular retention could potentially play a role in determining tumor remission and relapse.

Doxorubicin induces specific immune functions and cytokine expression in peritoneal cells.

To examine the basis of the immune modulation induced by the anticancer agent doxorubicin (DOX), the immunophenotype, tumoricidal activity, cytokine protein and mRNA expression were determined using peritoneal exudate cells (PEC) from saline-treated (untreated) and DOX-treated mice. A greater percentage of PEC from DOX-treated mice than from untreated mice were adherent to plastic, had characteristics of granulocytes, and were positive for the NK1.1, CD11b/Mac-1, and CD3 markers. DOX decreased the percentage of CD45R/B220+ cells. PEC from DOX-treated mice had greater tumoricidal potential than those from untreated mice since IL2, LPS, or IFNgamma alone increased the cytolytic activity of PEC from DOX-treated mice, whereas PEC from untreated mice required both LPS and IFNgamma to become cytolytic. DOX treatment modulated the expression of specific cytokines. Following stimulation in culture, PEC from DOX-treated mice produced more TNF, IL1, and IFNgamma than PEC from untreated mice. DOX treatment increased the levels of TNF, but not IL1, mRNA and decreased the levels of IL6 mRNA and protein. These data demonstrate that a single DOX injection induces specific effects in PEC and, as a consequence, increases the tumoricidal potential of cells of the macrophage and natural killer types.