Store-operated Ca²⁺entry (SOCE) is activated following depletion of the inositol-1,4,5-trisphosphate (InsP₃)-sensitive Ca²⁺pool to regulate proliferation in immortalized cell lines established from either primary or metastatic lesions. The molecular nature of SOCE may involve both Stim1, which senses Ca²⁺levels within the endoplasmic reticulum (ER) Ca²⁺reservoir, and a number of a Ca²⁺-permeable channels on the plasma membrane, including Orai1, Orai3, and members of the canonical transient receptor (TRPC1–7) family of ion channels. The present study was undertaken to assess whether SOCE is expressed and controls proliferation in primary cultures isolated from secondary lesions of heavily pretreated metastatic renal cell carcinoma (mRCC) patients. SOCE was induced following pharmacological depletion of the ER Ca²⁺store, but not by InsP₃-dependent Ca²⁺release. Metastatic RCC cells express Stim1-2, Orai1–3, and TRPC1–7 transcripts and proteins. In these cells, SOCE was insensitive to BTP-2, 10 µM Gd³⁺and Pyr6, while it was inhibited by 100 µM Gd³⁺, 2-APB, and carboxyamidotriazole (CAI). Neither Gd³⁺nor 2-APB or CAI impaired mRCC cell proliferation. Consistently, no detectable Ca²⁺signal was elicited by growth factor stimulation. Therefore, a functional SOCE is expressed but does not control proliferation of mRCC cells isolated from patients resistant to multikinase inhibitors.

Stim and Orai mediate constitutive Ca2+ entry and control endoplasmic reticulum Ca2+ refilling in primary cultures of colorectal carcinoma cells Stim and Orai mediate constitutive Ca2+ entry and control endoplasmic reticulum Ca2+ refilling in primary cultures of colorectal carcinoma cells

Nicotinic acid adenine dinucleotide phosphate (NAADP) gates two-pore channels 1 and 2 (TPC1 and TPC2) to elicit endo-lysosomal (EL) Ca²⁺ release. NAADP-induced EL Ca²⁺ signals may be amplified by the endoplasmic reticulum (ER) through the Ca²⁺-induced Ca²⁺ release mechanism (CICR). Herein, we aimed at assessing for the first time the role of EL Ca²⁺ signaling in primary cultures of human metastatic colorectal carcinoma (mCRC) by exploiting Ca²⁺ imaging and molecular biology techniques. The lysosomotropic agent, Gly-Phe β-naphthylamide (GPN), and nigericin, which dissipates the ΔpH which drives Ca²⁺ refilling of acidic organelles, caused massive Ca²⁺ release in the presence of a functional inositol-1,4,5-trisphosphate (InsP₃)-sensitive ER Ca²⁺ store. Liposomal delivery of NAADP induced a transient Ca²⁺ release that was reduced by GPN and NED-19, a selective TPC antagonist. Pharmacological and genetic manipulations revealed that the Ca²⁺ response to NAADP was triggered by TPC1, the most expressed TPC isoform in mCRC cells, and required ER-embedded InsP₃ receptors. Finally, NED-19 and genetic silencing of TPC1 reduced fetal calf serum-induced Ca²⁺ signals, proliferation, and extracellular signal-regulated kinase and Akt phoshorylation in mCRC cells. These data demonstrate that NAADP-gated TPC1 could be regarded as a novel target for alternative therapies to treat mCRC.

Treatment of advanced metastatic colorectal cancer (mCRC) patients is associated with a poor prognosis and significant morbidity. Moreover, targeted therapies such as anti-epidermal growth factor receptor (EGFR) have no effect in metastatic patients with tumors harboring a mutation in the RAS gene. The failure of conventional treatment to improve outcomes in mCRC patients has prompted the development of adoptive immunotherapy approaches including natural killer (NK)-based therapies. In this study, after confirmation that patients’ NK cells were not impaired in their cytotoxic activity, evaluated against long-term tumor cell lines, we evaluated their interactions with autologous mCRC cells. Molecular and phenotypical evaluation of mCRC cells, expanded in vitro from liver metastasis, showed that they expressed high levels of polio virus receptor and Nectin-2, whereas UL16-binding proteins were less expressed in all tumor samples evaluated. Two different patterns of MICA/B and HLA class I expression on the membrane of mCRC were documented; approximately half of mCRC patients expressed high levels of these molecules on the membrane surface, whereas, in the remaining, very low levels were documented. Resting NK cells were unable to display sizeable levels of cytotoxic activity against mCRC cells, whereas their cytotoxic activity was enhanced after overnight or 5-day incubation with IL-2 or IL-15. The susceptibility of NK-mediated mCRC lysis was further significantly enhanced after coating with cetuximab, irrespective of their RAS mutation and HLA class I expression. These data open perspectives for combined NK-based immunotherapy with anti-epidermal growth factor receptor antibodies in a cohort of mCRC patients with a poor prognosis refractory to conventional therapies.

Obtaining human tumor cell lines from fresh tumors is essential to advance our understanding of antitumor immune surveillance mechanisms and to develop new ex vivo strategies to generate an efficient anti-tumor response. The present study delineates a simple and rapid method for efficiently establishing primary cultures starting from tumor samples of different types, while maintaining the immuno-histochemical characteristics of the original tumor. We compared two different strategies to disaggregate tumor specimens. After short or long term in vitro expansion, cells analyzed for the presence of malignant cells demonstrated their neoplastic origin. Considering that tumor cells may be isolated in a closed system with high efficiency, we propose this methodology for the ex vivo expansion of tumor cells to be used to evaluate suitable new drugs or to generate tumor-specific cytotoxic T lymphocytes or vaccines.

Background The failure of conventional treatment and target therapies to significantly improve outcomes in metastatic colorectal cancer (mCRC) has prompted the development of immune-based therapies, including natural killer (NK) cells-based strategies. NK cells can kill target cells directly, as well as mediate antibody (Ab)-dependent cellular cytotoxicity (ADCC) via the membrane receptor FcgRIII, which binds to the Fc portion of IgG Ab. This feature makes them of particular importance in the immunotherapy of mCRC patients harboring the KRAS mutation that cannot benefit from administration of anti-epidermal growth factor receptor (EGFR) drugs. In this study we evaluated the capacity of patients derived NK cells, either resting or after cytokine activation, to lyse autologous mCRC cells. mCRC cells were analyzed for expression of ligands for adhesion and triggering NK receptors involved in their recognition and killing. We also evaluated whether KRAS mutated mCRC cells were susceptible to anti-EGFR-induced ADCC mediated by NK cells. Patients and methods. After obtaining informed signed consent, 25 mCRC patients have been enrolled to date. Tumor cells were disaggregated by GentleMACS Dissociator (Miltenyi Biotec,Germany), in vitro expanded and analyzed for the expression of ligands for NK triggering receptors. Ligands expression was evaluated by cytofluorimetric analysis and gene expression by qPCR on mCRC cultured cells and by immunohistochemistry in sections of samples embedded in paraffin. Resting and IL-2- or IL-15-activated NK cells, were analyzed for expression of triggering and inhibitory receptors and for their ability to kill autologous mCRC cells alone or after incubation with anti-EGFR monoclonal antibodies (mAbs) in a 51Cr release cytotoxicity assay. Results. Tumor cells were successfully expanded from 21 of 25 samples. Experiments performed in 10 patients showed the inability of patients resting NK cells to lyse mCRC cells (< 10% at effector:target ratio (E:T) of 20:1. Cytokine overnight (ON) activation resulted in an increased NK cytotoxic activity (IL-2: mean 28%; range:10-71; and IL-15: mean 40%; range 16-76 at E:T ratio of 20:1). Additional days of NK cell activation were able to further enhance their lytic capability. In resting NK cells the mean surface expression of activating receptors DNAM-1 and NKG2D was 79%, (range 75-91) and 39 % (range 29-58), respectively. The latter was up-regulated by ON cytokine activation (IL-2: mean 53%, range 48-70; IL-15: mean 70%, range 63-87). Among activating natural cytotoxicity receptors (NCRs), NKp46 is highly expressed both on resting and activated cells (>90%), while low surface expression of NKp30 and NKp44 was documented on resting NK cells (<10%). Their expression could be slightly up-regulated in particular after IL-15 ON activation (mean: 13%, range 9-30). Molecular analysis demonstrated a sizeable gene expression of NK ligands PVR, Nectin-2 and MICA/B on mCRC cells. Cytofluorimetric analysis showed that PVR and Nectin-2 are expressed in a vast majority of cultured mCRC cells (mean 65%, range 60-76; and mean 78%, range 70-98), respectively, while MICA/B were less expressed (mean 18%, range 15-25). The incubation of mCRC cells with anti-EGFR mAbs increased their susceptibility to NK-mediated lysis irrespective of KRAS status of tumor cells. The average rate of increase was greater using resting NK cells as effectors. The ongoing experiments comparing mCRC ligands expression evaluated on cultured tumor cells versus paraffin embedded sections will clarify whether results obtained in vitrocould be translated to the clinical setting. Conclusions. The evidence that ex vivo activated autologous NK cells are able to lyse patients tumor cells can offer new therapeutic options to a cohort of mCRC patients with a poor prognosis.