Small cell lung cancer: circulating tumor cell lines and expression of mediators of angiogenesis and coagulation.

Aim: Coagulation is frequently activated in cancer patients and has been correlated with an unfavorable prognosis. To evaluate whether a putative release of tissue factor (TF) by circulating tumor cells (CTCs) represents a target to impair the dissemination of small cell lung cancer (SCLC), the expression of relevant proteins in a panel of permanent SCLC and SCLC CTC cell lines that have been established at the Medical University of Vienna. Methods: Five CTC and SCLC lines were analyzed using a TF enzyme-linked immunosorbent assay (ELISA) tests, RNA sequencing, and western blot arrays covering 55 angiogenic mediators. Furthermore, the influence of topotecan and epirubicin as well as hypoxia-like conditions on the expression of these mediators was investigated. Results: The results demonstrate that the SCLC CTC cell lines express no significant amounts of active TF but thrombospondin-1 (TSP-1), urokinase-type plasminogen activator receptor (uPAR), vascular endothelial-derived growth factor (VEGF) and angiopoietin-2 in two cases. The major difference between the SCLC and SCLC CTC cell lines found was the loss of the expression of angiogenin in the blood-derived CTC lines. Topotecan and epirubicin decreased the expression of VEGF, whereas hypoxia-like conditions upregulated VEGF. Conclusions: Active TF capable of triggering coagulation seems not to be expressed in SCLC CTC cell lines in significant levels and, thus, CTC-derived TF seems dispensable for dissemination. Nevertheless, all CTC lines form large spheroids, termed tumorospheres, which may become trapped in clots of the microvasculature and extravasate in this supportive microenvironment. The contribution of clotting to the protection and dissemination of CTCs in SCLC may be different from other solid tumors such as breast cancer.

Cytotoxicity of combinations of the pan-KRAS SOS1 inhibitor BAY-293 against pancreatic cancer cell lines.

KRAS is mutated in approximately 25% of cancer patients and first KRAS G12C-specific inhibitors showed promising responses. Pancreatic cancer has the highest frequency of KRAS mutations but the prevailing KRAS G12D mutation is difficult to target. Inhibition of the GTP exchange factor (GEF) SOS1-KRAS interaction impairs oncogenic signaling independently of the specific KRAS mutations. In general, cell lines exhibiting KRAS mutations show specific alterations in respect to glucose utilization, signal transduction and stress survival. The aim of this investigation was to check the putative synergy of the SOS1 inhibitor BAY-293 with modulators targeting specific vulnerabilities of KRAS-mutated cell lines in vitro. The cytotoxicity of BAY-293 combinations was tested against MIA PaCa-2 (G12C), AsPC1 (G12D) and BxPC3 (KRAS wildtype) cell lines using MTT tests and calculation of the combination indices (CI) according to the Chou-Talalay method. The results show that BAY-293 synergizes with modulators of glucose utilization, inhibitors of the downstream MAPK pathway and several chemotherapeutics in dependence of the specific KRAS status of the cell lines. In particular, divergent responses for BAY-293 combinations between pancreatic and NSCLC cell lines were observed for linsitinib, superior inhibitory effects of trametinib and PD98059 in NSCLC, and lack of activity with doxorubicin in case of the pancreatic cell lines. Phosphoproteome analysis revealed inhibition of distinct signaling pathways by BAY-293 for MIA PaCa-2 on the one hand and for Aspc1 and BH1362 on the other hand. In conclusion, BAY-293 exhibits synergy with drugs in dependence of the tumor type and specific KRAS mutation.

Effects of metformin on human bone-derived mesenchymal stromal cell-breast cancer cell line interactions.

Metformin is used to treat patients with type 2 diabetes mellitus and was found to lower the incidence of cancer. Bone metastasis is a common impairment associated with advanced breast cancer. The present study investigated the effects of metformin on human bone-derived mesenchymal stromal cells (BM-MSC)-breast cancer cell line interactions. BM-MSCs grown from box chisels were tested for growth-stimulating and migration-controlling activity on four breast cancer cell lines either untreated or after pretreatment with metformin. Growth stimulation was tested in MTT tests and migration in scratch assays. Furthermore, the expression of adipokines of BM-MSCs in response to metformin was assessed using Western blot arrays. Compared to breast cancer cell lines (3.6 ± 1.4% reduction of proliferation), 500 µM metformin significantly inhibited the proliferation of BM-MSC lines (mean 12.3 ± 2.2 reduction). Pretreatment of BM-MSCs with metformin showed variable effects of the resulting conditioned media (CM) on breast cancer cell lines depending on the specific BM-MSC-cancer line combination. Metformin significantly reduced the migration of breast cancer cell lines MDA-MB-231 and MDA-MB-436 in response to CM of drug-pretreated BM-MSCs. Assessment of metformin-induced alterations in the expression of adipokines by BM-MSC CM indicated increased osteogenic signaling and possibly impairment of metastasis. In conclusion, the anticancer activities of metformin are the result of a range of direct and indirect mechanisms that lower tumor proliferation and progression. A lower metformin-induced protumor activity of BM-MSCs in the bone microenvironment seem to contribute to the positive effects of the drug in selected breast cancer patients.

Rovalpituzumab tesirine resistance: analysis of a corresponding small cell lung cancer and circulating tumor cell line pair.

Small cell lung cancer (SCLC) is frequently found disseminated at first presentation and holds a poor prognosis due to emerging resistance to first-line platinum-based and second-line topotecan chemotherapy. The present investigation tested the antitumor activity of rovalpituzumab tesirine (ROVA-T), a cytotoxic anti-DLL3 drug conjugate, against two SCLC and a corresponding SCLC CTC cell line established from a ROVA-T-resistant patient to characterize the mechanism of recurrence. Two cell lines were established from an SCLC patient progressing under ROVA-T therapy and characterized with respect to chemosensitivity against this drug as well as against currently applied chemotherapeutics and for their delta-like 3 (DLL3) expression. The chemosensitivity assays demonstrate that most SCLC lines show IC50 values exceeding the ROVA-T in-vivo concentrations and that slow-growing cells and lines showing spheroidal growth or proliferation as corresponding circulating tumor cells (CTCs) exhibit higher resistance. Chemosensitivity of the cell lines is not correlated with DLL3 protein expression possibly due to toxicity of the free payload in tissue culture. The clinical trials and experimental results demonstrate that refractoriness to ROVA-T is linked to a low initial tumor expression of DLL3, loss of DLL3 expression, higher chemoresistance to ROVA-T and the putative formation of resistant spheroids by the SCLC cells.

Synergistic cytotoxicity of the CDK4 inhibitor Fascaplysin in combination with EGFR inhibitor Afatinib against Non-small Cell Lung Cancer.

In the absence of suitable molecular markers, non-small cell lung cancer (NSCLC) patients have to be treated with chemotherapy with poor results at advanced stages. Therefore, the activity of the anticancer marine drug fascaplysin was tested against primary NSCLC cell lines established from pleural effusions. Cytotoxicity of the drug or combinations were determined using MTT assays and changes in intracellular phosphorylation by Western blot arrays. Fascaplysin revealed high cytotoxicity against NSCLC cells and exhibit an activity pattern different of the standard drug cisplatin. Furthermore, fascaplysin synergizes with the EGFR tyrosine kinase inhibitor (TKI) afatinib to yield a twofold increased antitumor effect. Interaction with the Chk1/2 inhibitor AZD7762 confirm the differential effects of fascplysin and cisplatin. Protein phosphorylation assays showed hypophosphorylation of Akt1/2/3 and ERK1/2 as well as hyperphosphorylation of stress response mediators of H1299 NSCLC cells. In conclusion, fascaplysin shows high cytotoxicity against pleural primary NSCLC lines that could be further boosted when combined with the EGFR TKI afatinib.

Cytotoxicity of combinations of the pan-KRAS inhibitor BAY-293 against primary non-small lung cancer cells.

KRAS is mutated in approximately 25% of Non-small Cell Lung Cancer (NSCLC) patients and first specific inhibitors showed promising responses that may be improved by concurrent interference with downstream signaling pathways. Cell lines exhibiting KRAS mutations show specific sensitivities to modulators affecting glucose utilization, signal transduction and cell survival. Novel SOS1-directed inhibitors with a broader anticancer coverage such as BAY-293 and BI-3406 inhibit KRAS through the hindrance of SOS1-KRAS interactions. The aim of this study was to check the putative synergy of BAY-293 with modulators having revealed specific vulnerabilities of KRAS-mutated cell lines. The present investigation tested the cytotoxicity of BAY-293 combinations against a series of Osimertinib-resistant primary NSCLC cell lines using MTT tests and calculation of combination indices according to the Chou-Talalay method. The results show that BAY-293 synergizes with modulators of glucose metabolism, inhibitors of cellular proliferation, several chemotherapeutics and a range of diverse modulators, thus corroborating the chemosensitivities of cells expressing mutated KRAS. In conclusion, BAY-293 exerts cytotoxicity with a wide range of drugs against Osimertinib-resistant primary NSCLC cell lines. The administration of pan-KRAS inhibitors alone may be limited in vivo by toxicity to normal tissues but made feasible by its use as part of suitable drug combinations. This study shows that BAY-293 combinations are active against NSCLC cells not further amenable to mutated EGFR-directed targeted therapy and results likewise hold relevance for pancreatic and colon cancer.

Cytotoxic activity of KRAS inhibitors in combination with chemotherapeutics.

INTRODUCTION: KRAS is the most frequently mutated oncogenic driver in pancreatic, lung, and colon cancer. Recently, KRAS inhibitors in clinical use show promising activity but most responses are partial and drug resistance develops. The use of therapeutics in combination with KRAS inhibitors are expected to improve outcomes. AREAS COVERED: This review describes the KRAS G12C mutation-specific inhibitors and the SOS1-targeting inhibitors that reduce the GTP-loading of wildtype and mutated KRAS. Both types of compounds reduce tumor cell proliferation in vitro and in vivo. The combinations of the various KRAS inhibitors with downstream signaling effectors, modulators of KRAS-associated metabolic alterations and chemotherapeutics are summarized. EXPERT OPINION: The clinical potency of mutated KRAS-specific inhibitors needs to be improved by suitable drug combinations. Inhibition of downstream signaling cascades increases toxicity and other combinations exploited comprise G12C-directed inhibitors with SOS1 inhibitors, glucose/glutamine metabolic modulators, classical chemotherapeutics, and others. The most suitable inhibitor combinations corroborated in preclinical development await clinical verification.

The Impact of NK Cell-Based Therapeutics for the Treatment of Lung Cancer for Biologics: Targets and Therapy.

Lung cancer has a dismal prognosis and novel targeted therapies leave still room for major improvements and better outcomes. Immunotherapy targeting immune checkpoint (IC) proteins, either as single agents or in combination with chemotherapy, is active but responders constitute only approximately 10-15% of non-small cell lung cancer (NSCLC) patients. Other effector immune cells such as CAR-T cells or NK cells may help to overcome the limitations of the IC inhibitor therapies for lung cancer. NK cells can kill tumor cells without previous priming and are present in the circulatory system and lymphoid organs. Tissue-residing NK cells differ from peripheral effector cells and, in case of the lung, comprise CD56bright CD16-negative populations showing high cytokine release but low cytotoxicity in contrast to the circulating CD56dim CD16-positive NK cells exhibiting high cytotoxic efficacy. This local attenuation of NK cell killing potency seems due to a specific stage of NK differentiation, immunosuppressive factors as well as presence of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (TREGs). Improved NK cell-based immunotherapies involve IL-2-stimulated effector cells, NK cells expanded with the help of cytokines, permanent NK cell lines, induced pluripotent stem cell-derived NK cells and NK cells armed with chimeric antigen receptors. Compared to CAR T cell therapy, NK cells administration is devoid of graft-versus-host disease (GvHD) and cytokine-release syndrome. Although NK cells are clearly active against lung cancer cells, the low-cytotoxicity differentiation state in lung tumors, the presence of immunosuppressive leucocyte populations, limited infiltration and adverse conditions of the microenvironment need to be overcome. This goal may be achieved in the future using large numbers of activated and armed NK cells as provided by novel methods in NK cell isolation, expansion and stimulation of cytotoxic activity, including combinations with monoclonal antibodies in antibody-dependent cytotoxicity (ADCC). This review discusses the basic characteristics of NK cells and the potential of NK cell preparations in cancer therapy.

Interactions of BRCA1-mutated Breast Cancer Cell Lines with Adipose-derived Stromal Cells (ADSCs).

Lipofilling may constitute a technique to assist reconstruction of breasts following prophylactic mastectomy for patients with mutated BRCA1 or BRCA2 genes. However, to date it is not clear whether adipose-derived stromal cells (ADSCs) increase the risk of tumor initiation and progression in this situation. Therefore, the interactions of BRCA1 mutated breast cancer cell lines with normal ADSCs were investigated in the present study. Characteristics of MDA-MB-436 (BRCA1 c.5277 + 1G > A) and HCC1937 (BRCA1 p.Gln1756.Profs*74) were compared to MDA-MB-231 and T47D BRCA1/2 wild-type breast cancer cell lines. ADSCs were cultivated from lipoaspirates of a panel of BRCA1/2- wildtype patients. Interactions of conditioned medium (CM) of these cells with the breast cancer lines were studied using proliferation and migration assays as well as adipokine expression western blot arrays. CM of ADSCs exhibit a dose-dependent stimulation of the proliferation of the breast cancer cell lines. However, of the ADSC preparations tested, only 1 out of 18 samples showed a significant higher stimulation of BRCA1-mutated MDA-MB-436 versus wildtype MDA-MB-231 cells, and all CM revealed lower stimulatory activity for BRCA1-mutated HCC1937 versus wildtype T47D cells. Additionally, migration of breast cancer cells in response to CM of ADSCs proved to be equivalent or slower for BRCA1/2 mutated versus nonmutated cancer cells and, with exception of angiopoietin-like 2, induced expression of adipokines showed no major difference. Effects of media conditioned by normal ADSCs showed largely comparable effects on BRCA1-mutated and wildtype breast cancer cell lines thus advocating lipofilling, preferentially employing allogeneic non-mutated ADSCs.

Effects of metformin on adipose-derived stromal cell (ADSC) - Breast cancer cell lines interaction.

AIMS: Metformin is a clinical drug administered to patients to treat type 2 diabetes mellitus that was found to be associated with a lower risk of occurrence of cancer and cancer-related death. The present study investigated the effects of metformin on human adipose-derived stromal cells (ADSC) - breast cancer cell line interactions. MAIN METHODS: ADSCs grown from lipoaspirates were tested for growth-stimulating and migration-controlling activity on breast cancer cell lines after pretreatment with metformin. Furthermore, secreted proteins of ADSCs, phosphorylation of intracellular proteins and the effect of metformin on adipocytic differentiation of ADSCs were assayed. KEY FINDINGS: Compared to breast cancer cell lines (4.0 ± 3.5% reduction of proliferation), 2 mM metformin significantly inhibited the proliferation of ADSC lines (19.2 ± 8.4% reduction of proliferation). This effect on ADSCs seems to be mediated by altered phosphorylation of GSK-3, CREB and PRAS40. Furthermore, treatment with metformin abolished the induction of differentiation of three ADSC lines to adipocytes. 1 and 2 mM metformin significantly impaired the migration of breast cancer cell lines MDA-MB-231 and MDA-MB-436 in scratch assays. SIGNIFICANCE: Metformin showed low direct inhibitory effects on breast cancer cell lines at physiological concentrations but exerted a significant retardation of the growth and the adipocytic differentiation of ADSCs. Thus, the anticancer activity of metformin in breast cancer at physiological drug concentrations seems to be mediated by an indirect mechanism that lowers the supportive activity of ADSCs.

Effects of salinomycin and niclosamide on small cell lung cancer and small cell lung cancer circulating tumor cell lines.

Tumor dissemination and recurrence is attributed to highly resistant cancer stem cells (CSCs) which may constitute a fraction of circulating tumor cells (CTCs). Small cell lung cancer (SCLC) constitutes a suitable model to investigate the relation of CTCs and CSCs due to rapid tumor spread and a high number of CTCs. Expansion of five SCLC CTC lines (BHGc7, 10, 16, 26 and UHGc5) in vitro at our institution allowed for the analysis of CSC markers and cytotoxicity of the CSC-selective drugs salinomycin and niclosamide against CTC single cell suspensions or CTC spheroids/ tumorospheres (TOS). Salinomycin exerted dose-dependent cytotoxicity against the SCLC lines but, with exception of BHGc7 TOS, there was no markedly enhanced activity against TOS. Similarly, niclosamide exhibits high activity against BHGc7 TOS and UHGc5 TOS but not against the other CTC spheroids. High expression of the CSC marker CD133 was restricted to three SCLC tumor lines and the BHGc10 CTC line. All SCLC CTCs are CD24-positive but lack expression of CD44 and ABCG2 in contrast to the SCLC tumor lines which show a phenotype more similar to that of CSCs. The stem cell marker SOX2 was found in all CTC lines and SCLC GLC14/16, whereas elevated expression of Oct-3/4 and Nanog was restricted to BHGc26 and UHGc5. In conclusion, the SCLC CTCs established from patients with relapsed disease lack a typical CSC phenotype in respect to chemosensitivity to CSC-selective drugs, surface markers, expression of pluripotent stem cell and transcription factors.

Non-small cell lung cancer-small cell lung cancer transformation as mechanism of resistance to tyrosine kinase inhibitors in lung cancer.

Mutated or rearranged driver kinases in non-small cell lung cancer (NSCLC) cells are clinically amenable to treatment with tyrosine kinase inhibitors (TKIs) resulting in prolonged survival and significant benefit compared to cytotoxic chemotherapy. The most frequent genomic alterations are observed for epidermal growth factor receptor and anaplastic lymphoma kinase, which can be blocked by a range of specific TKIs in sequence. In clinics, resistance to TKIs emerges after approximately one year and comprises secondary mutations of the kinases (on-target) or alternative pathways circumventing the original kinase (off-target) alterations. A special feature of NSCLC is the occurrence of histological transformation to small cell lung cancer (SCLC) in up to 14% of cases, which, in general, is accompanied by resistance to the original TKIs. SCLC transformed tumors may be treated with the classical platinum/etoposide regimen but thus far there are no definitive guidelines. Four transformed pleural SCLC lines in our lab indicate the presence of a gradual NSCLC-SCLC shift with overlapping drug sensitivities. In conclusion, the treatment of NSCLC-SCLC transformed cancer cells would need a better chemosensitivity assessment using functional genomics to guide further therapy.

Expression of Proteolytic Enzymes by Small Cell Lung Cancer Circulating Tumor Cell Lines.

Small cell lung cancer (SCLC) is an aggressive type of lung cancer which disseminates vigorously and has a dismal prognosis. Metastasis of SCLC is linked to an extremely high number of circulating tumor cells (CTCs), which form chemoresistant spheroids, termed tumorospheres. Intravasation and extravasation during tumor spread requires the activity of a number of proteases to disintegrate the stroma and vascular tissue. Generation of several permanent SCLC CTC lines allowed us to screen for the expression of 35 proteases using Western blot arrays. Cell culture supernatants of two CTC lines, namely BHGc7 and 10, were analyzed for secreted proteases, including matrix metalloproteinases (MMPs), ADAM/TS, cathepsins, kallikreins, and others, and compared to proteases expressed by SCLC cell lines (GLC14, GLC16, NCI-H526 and SCLC26A). In contrast to NCI-H526 and SCLC26A, MMP-9 was highly expressed in the two CTC lines and in GLC16 derived of a relapse. Furthermore, cathepsins (S, V, X/Z/P, A and D) were highly expressed in the CTC lines, whereas ADAM/TS and kallikreins were not detectable. In conclusion, SCLC CTCs express MMP-9 and a range of cathepsins for proteolysis and, aside from tissue degradation, these enzymes are involved in cell signaling, survival, and the chemoresistance of tumor cells.

Anticancer Activity of Fascaplysin against Lung Cancer Cell and Small Cell Lung Cancer Circulating Tumor Cell Lines.

Lung cancer is a leading cause of tumor-associated mortality. Fascaplysin, a bis-indole of a marine sponge, exhibit broad anticancer activity as specific CDK4 inhibitor among several other mechanisms, and is investigated as a drug to overcome chemoresistance after the failure of targeted agents or immunotherapy. The cytotoxic activity of fascaplysin was studied using lung cancer cell lines, primary Non-Small Cell Lung Cancer (NSCLC) and Small Cell Lung Cancer (SCLC) cells, as well as SCLC circulating tumor cell lines (CTCs). This compound exhibited high activity against SCLC cell lines (mean IC50 0.89 µM), as well as SCLC CTCs as single cells and in the form of tumorospheres (mean IC50 0.57 µM). NSCLC lines showed a mean IC50 of 1.15 µM for fascaplysin. Analysis of signal transduction mediators point to an ATM-triggered signaling cascade provoked by drug-induced DNA damage. Fascaplysin reveals at least an additive cytotoxic effect with cisplatin, which is the mainstay of lung cancer chemotherapy. In conclusion, fascaplysin shows high activity against lung cancer cell lines and spheroids of SCLC CTCs which are linked to the dismal prognosis of this tumor type. Derivatives of fascaplysin may constitute valuable new agents for the treatment of lung cancer.