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Immune checkpoint inhibitors (ICI) have improved outcomes for a variety of malignancies; however, many patients fail to benefit. While tumor-intrinsic mechanisms are likely involved in therapy resistance, it is unclear to what extent host genetic background influences response. To investigate this, we utilized the Diversity Outbred (DO) and Collaborative Cross (CC) mouse models. DO mice are an outbred stock generated by crossbreeding eight inbred founder strains, and CC mice are recombinant inbred mice generated from the same eight founders. We generated 207 DOB6F1 mice representing 48 DO dams and demonstrated that these mice reliably accept the C57BL/6-syngeneic B16F0 tumor and that host genetic background influences response to ICI. Genetic linkage analysis from 142 mice identified multiple regions including one within chromosome 13 that associated with therapeutic response. We utilized 6 CC strains bearing the positive (NZO) or negative (C57BL/6) driver genotype in this locus. We found that 2/3 of predicted responder CCB6F1 crosses show reproducible ICI response. The chromosome 13 locus contains the murine prolactin family, which is a known immunomodulating cytokine associated with various autoimmune disorders. To directly test whether prolactin influences ICI response rates, we implanted inbred C57BL/6 mice with subcutaneous slow-release prolactin pellets to induce mild hyperprolactinemia. Prolactin augmented ICI response against B16F0, with increased CD8 infiltration and 5/8 mice exhibiting slowed tumor growth relative to controls. This study highlights the role of host genetics in ICI response and supports the use of F1 crosses in the DO and CC mouse populations as powerful cancer immunotherapy models., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.)

This paper introduces a new mathematical model that is used to compute either the interfacial tension of quiescent axisymmetric pendant/sessile drops and pendant/captive bubbles. This model consists of the Young-Laplace equation, that describes interface shape, together with suitable boundary conditions that guarantee a prescribed volume of drops/bubbles and a fixed position in the capillary. In order to solve the problem numerically, the Young-Laplace equation is discretized by using numerical differentiation and the numerical solutions are obtained applying the well-know Newton method. The paper contains a validation of the new methodology presented for what theoretical bubble/drops are used. Finally, some numerical results are presented for both drops and bubbles of water as well as several surfactant solutions to demonstrate the applicability, versatility and reproducibility of the proposed methodology., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Fulvestrant is a dose dependent selective estrogen receptor (ER) down-regulator (SERD) used in ER-positive metastatic breast cancer (MBC). Nearly all patients develop resistance. We performed molecular analysis of circulating tumor cells (CTC) to gain insight into fulvestrant resistance. Preclinical studies were performed with cultured breast cancer cells spiked into human blood and analyzed on the CellSearch(®) system. Clinical data are limited to a subset of patients with ER-positive MBC from a previously reported pilot trial whose disease was progressing on fulvestrant (N = 7) or aromatase inhibitors (AIs) (N = 10). CTCs were enumerated and phenotyped for ER and B-cell lymphoma (BCL2) using the CellSearch(®) CXC kit. In preclinical modeling, tamoxifen and AIs resulted in stabilized ER expression, whereas fulvestrant eliminated it. Five of seven patients progressing on fulvestrant had ≥5CTC/7.5 ml WB. Two of these five, treated with 500 mg/month fulvestrant, had no detectable CTC-expression of ER and BCL2 (an ER regulated gene). Three patients had heterogeneous CTC-ER and BCL2 expression indicating incomplete degradation of the ER target by fulvestrant. Two of these patients received 250 mg/month whereas the third patient received 500 mg/month fulvestrant. Her cancer harbored a mutation (Y537S) in the estrogen receptor alpha gene (ESR1). All seven ER positive patients progressing on AIs had heterogeneous CTC-ER expression. These results suggest heterogeneous mechanisms of resistance to fulvestrant, including insufficient dosage, ESR1 mutation, or conversion to dependence on non-ER pathways. CTC enumeration, phenotyping, and genotyping might identify patients who would benefit from fulvestrant dose escalation versus switching to alternative therapies., (Copyright © 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Counterion competitive complexation is a background process currently ignored by using ionic hosts. Consequently, guest binding constants are strongly affected by the design of the titration experiments in such a way that the results are dependent on the guest concentration and on the presence of added salts, usually buffers. In the present manuscript we show that these experimental difficulties can be overcome by just considering the counterion competitive complexation. Moreover a single titration allows us to obtain not only the true binding constants but also the stoichiometry of the complex showing the formation of 1 : 1 : 1 (host : guest : counterion) complexes. The detection of high stoichiometry complexes is not restricted to a single titration experiment but also to a displacement assay where both competitive and competitive-cooperative complexation models are taken into consideration.

Purpose: Circulating tumor cells (CTC) are prognostic in metastatic breast cancer (MBC). We tested whether EpCAM-based capture system (CellSearch) is effective in patients with triple-negative (TN) MBC, and whether CTC apoptosis and clustering enhances the prognostic role of CTC., Experimental Design: CTC enumeration and apoptosis were determined using the CXC CellSearch kit at baseline and days 15 and 29 in blood drawn from TN MBC patients who participated in a prospective randomized phase II trial of nanoparticle albumin-bound paclitaxel (nab-paclitaxel) with or without tigatuzumab. Association between levels of CTC and patient outcomes was assessed using logistic regression, Kaplan-Meier curves, and Cox proportional hazards modeling., Results: Nineteen of 52 (36.5%), 14 of 52 (26.9%), and 13 of 49 (26.5%) patients who were evaluable had elevated CTC (≥5 CTC/7.5 mL whole blood) at baseline and at days 15 and 29, respectively. Patients with elevated versus not elevated CTC at each time point had worse progression-free survival (PFS; P = 0.005, 0.0003, 0.0002, respectively). The odds of clinical benefit response for those who had elevated versus low CTC at baseline and days 15 and 29 were 0.25 (95% CI: 0.08-0.84; P = 0.024), 0.19 (95% CI: 0.05-0.17; P = 0.014), and 0.06 (95% CI: 0.01-0.33; P = 0.001), respectively. There was no apparent prognostic effect comparing CTC apoptosis versus non-apoptosis. Presence of CTC cluster at day 15 and day 29 was associated with shorter PFS., Conclusions: CTC were detected using CellSearch assay in approximately one-third of TN MBC patients. Elevated CTC at baseline and days 15 and 29 were prognostic, and reductions in CTC levels reflected response., (©2015 American Association for Cancer Research.)

Background: Endocrine therapy (ET) fails to induce a response in one half of patients with hormone receptor (HR)-positive metastatic breast cancer (MBC), and almost all will eventually become refractory to ET. Circulating tumor cells (CTC) are associated with worse prognosis in patients with MBC, but enumeration alone is insufficient to predict the absolute odds of benefit from any therapy, including ET. We developed a multiparameter CTC-Endocrine Therapy Index (CTC-ETI), which we hypothesize may predict resistance to ET in patients with HR-positive MBC., Methods: The CTC-ETI combines enumeration and CTC expression of four markers: estrogen receptor (ER), B-cell lymphoma 2 (BCL-2), Human Epidermal Growth Factor Receptor 2 (HER2), and Ki67. The CellSearch System and reagents were used to capture CTC and measure protein expression by immunofluorescent staining on CTC., Results: The feasibility of determining CTC-ETI was initially established in vitro and then in a prospective single-institution pilot study in patients with MBC. CTC-ETI was successfully determined in 44 of 50 (88%) patients. Eighteen (41%), 9 (20%), and 17 (39%) patients had low, intermediate, and high CTC-ETI scores, respectively. Interobserver concordance of CTC-ETI determination was from 94% to 95% (Kappa statistic, 0.90-0.91). Inter- and cell-to-cell intrapatient heterogeneity of expression of each of the CTC markers was observed. CTC biomarker expression was discordant from both primary and metastatic tissues., Conclusions: CTC expression of ER, BCL-2, HER2, and Ki67 can be reproducibly measured with high analytical validity using the CellSearch System. The clinical implications of CTC-ETI, and of the heterogeneity of CTC biomarker expression, are being evaluated in an ongoing prospective trial., (©2014 American Association for Cancer Research.)

Urine is a suitable biological fluid to look for markers of physiological and pathological processes, including renal and nonrenal diseases. In addition, it is an optimal body sample for diagnosis, because it is easily obtained without invasive procedures and can be sampled in large quantities at almost any time. Rats are frequently used as a model to study human diseases, and rat urine has been analyzed to search for disease biomarkers. The normal human urinary proteome has been studied extensively, but the normal rat urinary proteome has not been studied in such depth. In light of this, we were prompted to analyze the normal rat urinary proteome using three complementary proteomics platforms: SDS-PAGE separation, followed by LC-ESI-MS/MS; 2DE, followed by MALDI-TOF-TOF and 2D-liquid chromatography-chromatofocusing, followed by LC-ESI-Q-TOF. A total of 366 unique proteins were identified, of which only 5.2% of unique proteins were identified jointly by the three proteomics platforms used. This suggests that simultaneous proteomics techniques provide complementary and nonredundant information. Our analysis affords the most extensive rat urinary protein database currently available and this may be useful in the study of renal physiology and in the search for biomarkers related to renal and nonrenal diseases., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: The methods routinely used for bacterial identification in Clinical Microbiology Laboratory, although miniaturized and automated, are still based on the same basic principles as classical identification methods. Nevertheless, technological advances are emerging which could modify these routine methods. We report a comparative study between conventional identification methods and mass spectrometry MALDI-TOF (MS MALDI-TOF) for bacterial identification in the Clinical Microbiology Laboratory., Methods: We analysed 294 facultative anaerobic and aerobic isolates (65 Gram positives and 229 Gram negatives), obtained from different clinical samples, using conventional microbiological methods (Wider, Fco. Soria Melguizo, Madrid, Spain; Vitek-2, APIStaph, API 20 Strep, API Coryne and API NH, bioMérieux, Marcy L'Etoile, France) and an Autoflex III MS with a MALDI-TOF device (Bruker Daltonics GmbH, Leipzig, Germany). Salmonella isolates were also typed by using specific sera. Isolates identified with a confidence rate <95% were checked by using API systems. Isolates which were not accurately identified by API systems were rejected. MS MALDI-TOF identification is automatically scored by the system software between 1 and 3 points. Isolates with scores <1.5 were classified as unreliable. Correlation between both identifications was classified as correlation at the species level, at the genus level or no correlation., Results: Correlation at the species level in Gram positives was 100%. Correlation in Gram negatives was 87.7% at the species level and 97.7% at the genus level. There was no correlation in 2.2% of Gram negatives studied. Identification failures occurred in the genera Raoultella and Acinetobacter, in Stenotrophomonas maltophilia and in Francisella tularensis., Conclusion: Bacterial clinical isolates identification obtained by MS MALDI-TOF shows excellent correlation with identification obtained by conventional microbiological methods. Moreover, MS MALDI-TOF allows the identification of bacteria from colonies grown on agar culture plates in just a few minutes, with a very simple methodology and hardly any consumable costs, although the financial costs of purchasing the device can be high., (Copyright © 2009 Elsevier España, S.L. All rights reserved.)

Protein analysis in biological fluids has been used for many years for diagnosis and monitoring of diseases. First it was quantification of total protein, afterwards the electrophoretic separation of proteins and later the quantification of specific proteins using immunoassays. These proteins are used as biological markers (biomarkers) of disease. Since a few years, proteomics allows the simultaneous analysis of hundreds of proteins, as well as the analysis of their structural modifications. In this review the current situation of clinical proteomics for the discovery of new biomarkers in biological fluids is presented.