Sertoli cells from mammalian testis are key cells involved in development and maintenance of stem cell spermatogonia as well as secretion of a chloride-and potassium-rich fluid into the lumen of seminiferous tubules. Using whole-cell patch clamp experiments, a novel chloride current was identified. It is activated only in the presence of an extracellular acidic pH, with an estimated half-maximal activation at pH 5.5. The current is strongly outwardly rectifying, activated with a fast time-dependent onset of activation but a slow timedependent kinetic at depolarization pulses. The pH-activated chloride current was not detected at physiological or basic pH and is not sensitive to intracellular or extracellular Ca 2؉ variation. Diphenylamine-2-carboxylic acid and 4,4-diisothiocyanatostilbene-2,2-disulfonic acid blocked the induced currents, and its anionic selectivity sequence was Cl ؊ > Br ؊ > I ؊ > gluconate. We have performed a reverse transcription-PCR analysis to search for voltage-dependent chloride rClC channels in cultured rat Sertoli cells. Among the nine members of the family only rClC-2, rClC-3, rClC-6, and rClC-7 have been identified. The inwardly rectifying rClC-2 chloride current was activated by hyperpolarization but not by pH variation. A different depolarization-activated outwardly rectifying chloride current was activated only by hypotonic challenge and may correspond either to rClC-3 or rClC-6. Immunolocalization experiments demonstrate that rClC-7 resides in the intracellular compartment of Sertoli cells. This study provides the first functional identification of a native acid-activated chloride current. Based on our molecular analysis of rClC proteins, this new chloride current does not correspond to rClC-2, rClC-3, rClC-6, or rClC-7 channels. The potential physiological role of this native current in an epithelial cell from the reproductive system is discussed.

Purpose: Regorafenib is synergistic with immune checkpoint inhibition in colorectal cancer preclinical models.Patients and Methods: This was a single-arm, multicentric phase II trial. Regorafenib was given 3 weeks on/1 week off, 160 mg every day; avelumab 10 mg/kg i.v. was given every 2 weeks, beginning at cycle 1, day 15 until progression or unacceptable toxicity. The primary endpoint was the confirmed objective response rate under treatment, as per RECIST 1.1. The secondary endpoints included a 1-year nonprogression rate, progression-free survival (PFS), and overall survival (OS), safety and biomarkers studies performed on sequential tumor samples obtained at baseline and at cycle 2 day 1.Results: Forty-eight patients were enrolled in four centers. Fortythree were assessable for efficacy after central radiological review. Best response was stable disease for 23 patients (53.5%) and progressive disease for 17 patients (39.5%). The median PFS and OS were 3.6 months [95% confidence interval (CI), 1.8-5.4] and 10.8 months (95% CI, 5.9-NA), respectively. The most common grade 3 or 4 adverse events were palmar-plantar erythrodysesthesia syndrome (n ¼ 14, 30%), hypertension (n ¼ 11, 23%), and diarrhea (n ¼ 6, 13%). High baseline infiltration by tumor-associated macrophages was significantly associated with adverse PFS (1.8 vs. 3.7 months; P ¼ 0.002) and OS (3.7 months vs. not reached; P ¼ 0.002). Increased tumor infiltration by CD8 þ T cells at cycle 2, day 1 as compared with baseline was significantly associated with better outcome.Conclusions: The combination of regorafenib þ avelumab mobilizes antitumor immunity in a subset of patients with microsatellite stable colorectal cancer. Computational pathology through quantification of immune cell infiltration may improve patient selection for further studies investigating this approach.

BackgroundTopoisomerase I (TOP1) is a nuclear enzyme that catalyzes the relaxation of supercoiled DNA during DNA replication and transcription. TOP1 is the molecular target of camptothecin and related drugs such as irinotecan and SN38 (irinotecan's active metabolite). Irinotecan is widely used as an anti-cancer agent in the treatment of metastatic colon cancer. However, its efficacy is often limited by the development of resistance.MethodsWe previously established several SN38 resistant HCT116-derived clones to study the mechanisms underlying resistance to SN38. Here, we investigated whether resistance to SN38 in these cell lines could be linked to the presence of TOP1 mutations and changes in its expression and activity. Functional analyses were performed on these cell lines challenged with SN38 and we specifically monitored the double strands breaks with γH2AX staining and replication activity with molecular combing.ResultsIn SN38 resistant HCT116 clones we identified three new TOP1 mutations, which are located in the core subdomain III (p.R621H and p.L617I) and in the linker domain (p.E710G) and are packed together at the interface between these two domains. The presence of these TOP1 mutations in SN38 resistant HCT116 cells did not modify TOP1 expression or intrinsic activity. Conversely, following challenge with SN38, we observed a decrease of TOP1-DNA cleavage complexes and a reduction in double-stranded break formation). In addition, we showed that SN38 resistant HCT116 cells present a strong decrease in the SN38-dependent asymmetry of replication forks that is characteristic of SN38 sensitive HCT116 cells.ConclusionsThese results indicate that the TOP1 mutations are involved in the development of SN38 resistance. We hypothesize that p.L617, p.R621 and p.E710 TOP1 residues are important for the functionality of the linker and that mutation of one of these residues is sufficient to alter or modulate its flexibility. Consequently, linker fluctuations could have an impact on SN38 binding by reducing the enzyme affinity for the drug.