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The hepatocyte growth factor (HGF) receptor MET is an oncogenic driver in a subpopulation of Non-small Lung Cancer Cells (NSCLC) at the primary tumor stage or in acquired resistance to treatment with tumor-targeting tyrosine kinase inhibitors (TKIs).This article summarizes the mechanisms leading to overexpression and activation of MET by amplification and mutations including exon 14 aberrations. Furthermore, the methods to detect and categorize MET as a tumor driver and the selective TKIs for patient treatment are discussed.Activating mutations and rearrangements of kinases in NSCLC are the target of successful therapeutic intervention. However, MET activation involves a number of complex alterations including gene amplification, prevention of degradation by METex14 exon skipping and a host of gene mutations. A high-level MET expression is the precondition for tumor responses to TKIs and the confirmation of MET-dependent tumor progression is difficult in primary lesions and in tumors exhibiting resistance to mutated EGFR-directed therapy in absence of standardized and concordant assays of MET amplification.

Abstract: The most frequent mutated oncogene KRAS in lung cancer is targeted by KRAS G12C-directed drugs, such as Sotorasib and Adagrasib. Still, other alleles frequently expressed in pancreatic and colon cancer may be attacked indirectly by hitting the guanine nucleotide exchange factor (GEF) SOS1 that loads and activates KRAS. The first modulators of SOS1 were found to act as agonists and defined as a hydrophobic pocket at the catalytic site. High throughput screenings resulted in the detection of SOS1 inhibitors Bay-293 and BI-3406 comprising amino quinazoline scaffolds optimized for binding to the pocket by various substituents. The first inhibitor, BI-1701963, is in clinical studies alone or in combination with a KRAS inhibitor, a MAPK inhibitor or chemotherapeutics. An optimized agonist, VUBI-1, shows activity against tumor cells by destructive overactivation of cellular signaling. This agonist was used to formulate proteolysis targeting chimera (PROTAC), that labels SOS1 for degradation by proteasomal degradation through a linked VHL E3 ligase ligand. This PROTAC exhibited the highest SOS1-directed activity due to target destruction, recycling and removal of SOS1 as a scaffolding protein. Although other first PROTACs have entered clinical trials, each conjugate must be meticulously adapted as an efficient clinical drug.

Lung cancer is the most prevalent type of cancer globally and small cell lung cancer (SCLC) accounts for only 15% of all cases but exhibits a dismal prognosis. The standard of care of SCLC has not changed for decades and novel biomarkers and novel strategies for patient's care are urgently needed.The expression of the two potential markers MUC1 and CD147 was evaluated in circulating tumor cells (CTCs) and CTC-derived SCLC cell lines using qRT PCR, western blotting, immunohistochemistry, and ELISA assays.Both CTCs enriched from patient blood samples by Parsortix isolation technology and SCLC/CTC cell lines exhibited significant expression of MUC1 and CD147. Silencing of MUC1 increased chemosensitivity of an SCLC line to topotecan.Both markers, MUC1 and CD147, are highly expressed in patient-derived SCLC and SCLC CTC cell lines and show promise as potential biomarkers in SCLC.

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.

SummaryIn 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.

Introduction : Mesenchymal stromal/stem cells (MSCs) hold great perspective for the therapy of a host of diseases due to regenerative and anti-inflammatory properties by differentiation into diverse cell populations, homing to damaged tissue regions, paracrine effects and release of extracellular vesicles. Areas covered : This review describes the isolation, characterization and potential use of MSCs and ADSCs for benign and malignant diseases. The MSCs may be administered as whole cells or in form of their secretome that is held responsible for most of their beneficial effects. A special constituent of the paracrine components are the extracellular vesicles (EVs) that carry a biologically potent cargo of proteins, cytokines and RNA. Expert opinion : The applications of MSCs and ADSCs are amply documented and have been investigated in preclinical models and many unregulated and a few controlled trials. Larger numbers of MSCs and ADSCs can be obtained for allogeneic transfer but imply difficulties including perseverance of the cells in vivo and possible differentiation into harmful cell types. MSC-derived cell-free preparations are easier to handle and manufacture for various applications. Especially, with the help of bioreactors, EVs can be obtained in excessive numbers and preloaded or charged with proteins, cytokines and regulatory RNA specimen to treat inflammatory diseases and cancer.

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.

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.

Immune checkpoint inhibition (ICI) has proved successful for selected tumors and a subpopulation of patients. The human monoclonal IgG1 antibody (mAB) avelumab capable of mediating antibody-dependent cytotoxicity (ADCC) lysis is directed to programmed death ligand-1 (PD-L1) of tumor cells and is tested in trials aiming to improve ICI in combination with chemotherapeutic drugs.This article presents an overview of the current trials to enhance ICI regimens using avelumab in combination with chemotherapeutics, antiangiogenetic drugs, and immunomodulators. Predictive factors for this kind of immunochemotherapy are discussed.Clinical data demonstrate that avelumab shows efficacy in cancer patients against Merkel cell carcinoma (MCC), renal cell carcinoma (RCC), and urothelial cancers as single agent. Furthermore, avelumab in combination with axitinib in RCC increases survival and exhibits activity in combination with docetaxel in urothelial carcinoma. However, several other immunochemotherapy trials for ovarian cancer, gastric cancer, and non-small lung cancer (NSCLC) showed no activity due to factors disfavoring administration of immunotherapy combos.

Introduction: Coagulation is frequently activated in cancer patients and has been correlated with an unfavorable prognosis. To evaluate whether a release of tissue factor (TF) of circulating tumor cells (CTCs) is involved in the dissemination of small cell lung cancer (SCLC) we studied the expression of relevant proteins in a panel of permanent SCLC and SCLC CTC cell lines (BHGc7, BHGc10, BHGc16, BHGc26 and UHGc5) that have been established at our institution. Material and Methods Five CTC and SCLC lines were analyzed using Western blot arrays covering 55 angiogenetic mediators. Furthermore, the influence of topotecan and epirubicin as well as hypoxia-like conditions on the expression of these mediators and cells was investigated. Results The results demonstrate that the SCLC lines express no significant amounts of the TF but thrombospondin-1 (TSP-1), uPAR, 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 expression of VEGF, whereas hypoxia-like conditions upregulated VEGF. Conclusions Active TF seems not to be expressed in SCLC and 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 protection and dissemination of CTCs in SCLC seems to be different from other solid tumors such as breast cancer.

BACKGROUND AND OBJECTIVE: Small cell lung cancer (SCLC) and Ewing’s sarcoma (ES) at the disseminated stage are not amenable to therapy and have a dismal prognosis with low survival rates. Despite representing different tumor entities, treatment for both malignancies relies on cytotoxic chemotherapy that has not considerably changed for the past decades. The genomic background has been extensively studied and found to comprise inactivation of p53 and RB1 in case of SCLC and EWSR1/FLI1 rearrangement in case of ES resulting in aggressive tumors in adults with heavy tobacco consumption and as bone tumor in juveniles, respectively. New therapeutic modalities are urgently needed to improve the outcomes of both tumor entities, especially in patients with metastatic disease or recurrences. This review summarizes the common cell biologic and clinical characteristics of difficult-to-treat SCLC and ES and discusses their refractoriness and options to improve the therapeutic efficacy. METHODS: PubMed and Euro PMC were searched from January 1(st), 2012 to January 16(th), 2022 using the following key words: “SCLC”, “Ewing´s sarcoma”, “Genomics” and “Chemoresistance” as well as own work. KEY CONTENT AND FINDINGS: Therapy of SCLC and ES involves the use of undirected cytotoxic drugs in multimodal chemotherapy and administration of topotecan for 2(nd) line SCLC regimens. Despite highly aggressive chemotherapies, outcomes are dismal for patients with disseminated tumors. A host of unrelated drugs and targeted therapeutics have failed to result in progress for the patients and the underlying mechanisms of chemoresistance are still not clear. Identification of chemoresistance-reversing modulators in vitro and patient-derived xenografts of SCLC and ES has not translated into new therapies. CONCLUSIONS: The global chemoresistance of SCLC and ES may be explained by physiological resistance at the tumor level and formation of larger spheroids that contain quiescent and hypoxic tumor cells in regions that occlude therapeutics. This type of chemoresistance is difficult to overcome and prevent the accumulation of effective drug concentration at the tumor cell level to a significant degree leaving therapeutic interventions of any kind ineffective.

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 complication of 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 (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 impaired 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 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.

SummaryTumor 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.

Small cell lung cancer (SCLC) is a highly malignant disease with a dismal prognosis that is currently being tested for theclinical activity of checkpoint inhibitors. SCLC is associated with smoking and exhibits a high mutational burden. However, low expression of PD-L1 and MHC antigens, as well low levels of immune cell infiltration and rapid tumor progress seems to limit the efficacy of anticancer immunity. Nevertheless, long-term survival was reported from studies using anti-PD-1/PD-L1 and CTLA-4 agents.Data of clinical trials of checkpoint inhibitors in SCLC show lower success rates compared to NSCLC. The mechanisms of resistance to immunotherapy are discussed for their relevance to SCLC patients.Although some factors, such as a high mutation rate, favor immunotherapy for SCLC patients, downregulation of MHC class I, low expression of PD-L1, poor tumor infiltration by effector T cells, presence of myeloid-derived suppressor cells as well as regulatory T lymphocytes counteract the immune system activation by checkpoint inhibitors. Furthermore, this tumor develops avascular regions which have immunosuppressive effects and restrict access of lymphocytes and antibodies. In conclusion, immunotherapy in SCLC is effective in highly selected patients with good performance status and special and unknown preconditions contributing to long-lasting responses.

Metformin is used to treat patients with diabetes mellitus and that was found to lower the incidence of cancer. The present study investigated the effects of metformin on human bone-derived mesenchymal stromal cells (BM-MSC) and their breast cancer cell line interactions. BM-MSCs were tested for growth stimulation and migration controlling activity on four breast cancer cell lines employing MTT tests, migration scratch tests and assays of the expression of adipokines in Western Blot arrays. Compared to breast cancer cell lines, metformin significantly inhibited the proliferation of BM-MSC lines. Pretreatment of BM-MSCs with metformin showed variable effects on breast cancer cell lines depending on the specific BM-MSC cancer line combination. Metformin significantly impaired the migration of MDA-MB-231 and MDA-MB-436 in response to conditioned media (CM) of drug pretreated BM-MSCs. Metformin-induced alterations of adipokines by BM-MSC CM indicated increased osteogenic signaling and possibly impairment of metastasis. The anticancer activities of metformin seem to be the result of direct and indirect mechanisms. A lower metformin-induced protumor activity of BM-MSCs in the bone microenvironment seem to contribute to the anticancer effects of this drug in breast cancer patients.

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.

Introduction In-depth genomic characterization of thymic epithelial tumors (TETs), comprising thymomas and thymic carcinomas (TCs), failed to identify targetable mutations and suggested unique biology of TETs, including KIT expression in most TCs. Recently, tuft cell–like medullary thymic epithelial cells were identified in the murine thymus, and our reanalysis of the published gene expression data revealed that these cells express KIT. In addition, recently, a minor subset of SCLCs with tuft cell–like features was described. Methods We interrogated mRNA expression data from our tumor cohorts (N = 60) and publicly available, independent data sets from TETs and NSCLC (N = 1199) for expression of tuft cell genes and KIT. Expression of KIT and of POU2F3 protein, the master regulator of tuft cells, was analyzed in cancer tissue (N = 344) by immunohistochemistry. Results Normal human thymic tuft cells and most TCs coexpressed KIT and known tuft cell genes, particularly POU2F3 and GFI1B. Unexpectedly, small subsets of tuft cell–like tumors coexpressing POU2F3, GFI1B, and KIT were also identified among pulmonary squamous cell carcinomas, adenocarcinomas, and large cell neuroendocrine carcinoma and clustered together in each histologic cohort. In addition to the tuft cell–like signature, both thymic and lung tuft cell–like carcinomas had distinct genetic, pathologic, and clinical features in each cohort. Conclusions We suggest that the tuft cell–like phenotype defines novel subsets of thymic and pulmonary carcinoma. Its high prevalence in thymic squamous cell carcinomas that have no known toxic or viral etiologies suggests a new mechanism of carcinogenesis that may lead to specific drug susceptibilities.

Introduction: Drug screening assays employing two-dimensional (2D) cultures of cancer cells have been largely replaced by three-dimensional (3D) multicellular tumor spheroid (MCTS) models which more closely represent patient's tumors. The predictive power of the different MCTSs depends on source of the cells, techniques of preparation, and characteristics of the aggregates. Areas covered: The preparation of MCTSs and a comparison of the spheroids assembled from permanent cancer and patient-derived cell lines in respect to the correlation of their chemosensitivity to clinical responses are discussed. Spheroids formed in in vivo in pleural effusion and blood of cancer patients are presented as interesting sources for drug screening. Expert opinion: 3D tumor models for drug screening were adopted to increase the predictive power of assays for success in clinical trials. Cell lines which form dense spheroids differ in physical properties, gene expression, and chemosensitivity from 2D cultures. Still, most of these MCTS models lack characteristics of complex tumor tissues and have not been validated for their adequacy to select clinically useful drugs. Patient-derived spheroids from pleural effusion or blood, namely tumorospheres of circulating tumor cells, are MCTS models most similar to patient's tumors.

The efficacy of platinum-based chemotherapy for patients with non-small cell lung cancer (NSCLC) is limited by chemoresistance. Platinum drugs damage DNA by introducing intrastrand and interstrand crosslinks which result in cell death. Excision repair cross-complementing 1 (ERCC1) is a member of the nucleotide excision repair (NER) pathway which erases such defects. Single nucleotide polymorphisms (SNPs) in ERCC1 impair this activity and have been suggested to predict the response to chemotherapy. Area covered: Among the polymorphisms of proteins involved in uptake, metabolism, cytotoxicity and efflux of platinum drugs, codon 118 C/T and C8092A in ERCC1 are the best characterized SNPs studied for their predictive power. Here, the divergent results for studies of these markers in NSCLC are summarized and the reasons for this contradictory data discussed. Expert opinion: Cytotoxicity of platinum compounds comprise complex cellular processes for which DNA repair may not constitute the rate limiting step. These drugs are administered as doublets to histologically diverse patients and, furthermore, the NER pathway in ERCC1 wildtype cohorts may be still impaired by the chemotherapeutics applied. At present, assessment of a limited number of polymorphism in DNA repair proteins is not reliably associated with response to treatment in NSCLC patients.

Small cell lung cancer (SCLC) represents 15% of lung cancers and is characterized by early dissemination, development of chemoresistance and a poor prognosis. A host of diverse drugs failed invariably and its mechanisms of global chemoresistance have not been characterized so far. SCLC represents the prototype of an aggressive and highly metastatic tumor which is ultimately refractory to any treatment. High numbers of circulating tumor cells (CTCs) allowed us to establish 5 CTC cell lines (BHGc7, 10, 16, 26 and UHGc5) from patients with recurrent SCLC. These cell lines exhibit the typical SCLC markers and CTCs of all patients developed spontaneously large multicellular aggregates, termed tumorospheres. Ki67 and carbonic anhydrase 9 (CAIX) staining of tumorosphere sections revealed quiescent and hypoxic cells, respectively. Accordingly, comparison of the chemosensitivity of CTC single cell suspensions with tumorospheres demonstrated increased resistance of the clusters against chemotherapeutics commonly used for treatment of SCLC. Therefore, global chemoresistance of relapsing SCLC seems to rely on formation of large tumorospheres which reveal limited accessibility, lower growth fraction and hypoxic conditions. Since similar tumor spheroids were found in other tumor types, SCLC seems to represent a unique tumor model to study the association of CTCs, metastasis and drug resistance.

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.

Background Assisted lipotransfer for breast reconstruction involves the isolation and supplementation of adipose-derived stromal cells. This procedure has raised concerns regarding safety with respect to promotion of tumor growth and relapse. Several in vitro and animal experimental studies have indicated increased survival, growth, and invasive characteristics of breast cancer cells on interaction with adipose-derived stromal cells. These results seem to be in poor concordance with clinical observations of a low rate of cancer recurrences after assisted lipotransfer. Methods The authors investigated the effects of adipose-derived stromal cells and adipose-derived stromal cells differentiated into adipocytes and fibroblasts on five breast cancer cell lines (i.e., T47D, MCF-7, BT20, MDA-MB-231, and ZR-75-1) and MCF-10A, a nonmalignant counterpart. Results Conditioned media of adipose-derived stromal cells stimulated the proliferation of breast cancer cell lines depending on the individual adipose-derived stromal cell-breast cancer cell line combination. Conditioned media of adipose-derived stromal cells differentiated into adipocytes gave a lower response, and conditioned media of fibroblasts were also active. A putative cancer stem cell-like phenotype was not increased by adipose-derived stromal cell-conditioned media, no physical interaction of cancer cells with adipose-derived stromal cells was detectable on scanning electron microscopy, and cell migration was not enhanced. Adipogenic differentiation of adipose-derived stromal cells indicated that hepatocyte growth factor, insulin-like growth factor-binding protein-3, insulin-like growth factor-binding protein-6, interleukin-6, CCL2/MCP-1, and macrophage colony-stimulating factor are not linked to the proliferative activity of conditioned media. Conclusion The results indicate that the adipose-derived stromal cells used for assisted lipotransfer are not expected to increase the risk of tumor recurrence to a major degree in correspondence with the clinical observation of the affected breast cancer patients. Clinical question/level of evidence Therapeutic, V.

Introduction: Brigatinib is a second-line inhibitor for the treatment of rearranged anaplastic lymphoma kinase (ALK) in lung cancer patients which has significant activity against brain metastases. This tyrosine kinase inhibitor (TKI) overcomes a wide range of ALK mutations which confer therapeutic resistance and is increasingly applied in first-line therapy due to improved benefit for patients compared to crizotinib, the current standard of care. Areas covered: The authors review the development and characteristics of brigatinib and discuss the optimal clinical use and sequence of the application of ALK inhibitors in patients progressing under therapy. Expert opinion: ALK-rearranged NSCLC can be treated with a broad range of approved and novel inhibitors at various stages of therapy, including the second-line therapeutic brigatinib. Besides this TKI, the second-line ALK inhibitors alectinib and ceritinib, as well as the third-line lorlatinib are approved for the treatment of ALK-positive NSCLC patients. The main challenge is to find sequences and combinations of ALK inhibitors which provide the best benefit for the patients.

Gastric cancer exhibits a poor prognosis and is the third most common cause of cancer death worldwide. Chemotherapy of metastatic gastric cancer is based on combinations of platinum drugs and fluoropyrimidines, with added agents. Oxoplatin is a stable oral platinum(IV) prodrug which is converted to a highly active tetrachlorido(IV) complex under acidic conditions. In the present work, we studied the cytotoxic effects of oxoplatin against a panel of four gastric cancer cell lines in vitro. Furthermore, the role of HSP90 in chemoresistance of these lines was investigated using the specific inhibitor ganetespib. The KATO-III, MKN-1, MKN-28, MKN-45 lines were used in MTT chemosensitivity, cell cycle and apoptosis assays. KATO-III is a signet ring diffuse cell type, MKN-1 an adenosquamous primary, MKN-28 a well-differentiated intestinal type and the MKN-45 a poorly differentiated, diffuse type gastric carcinoma line. Cytotoxicity was tested in MTT assays and intracellular signal transduction with proteome profiler Western blot arrays. Interactions of platinum drugs and ganetespib were calculated with help of the Chou-Talalay method. The prodrug oxoplatin revealed low activity against the four gastric cancer cell lines, whereas the platinum tetrachlorido(IV) complex and cisplatin gave IC50 values of 1-3 µg/ml with increasing chemoresistance observed in the order of MKN-1, KATO-III, MKN-28 to MKN-45. With exception of KATO-III and MKN-28/oxoplatin, all other cell lines featured marked synergistic toxicity with clinically achievable concentrations of ganetespib. Oral administration of a platinum agent such as oxoplatin would be of great value for patients and care providers alike. These results suggest that the oncogene-stabilizing HSP90 chaperone represents an important mediator of chemoresistance in gastric cancer. Ganetespib reduced the phosphorylation of p53, Akt1/2/3 and PRAS40, as well as of WNK1, a kinase which regulates intracellular chloride concentrations. Intracellular chloride was reported to control proliferation of gastric cancer cell lines. Expression of MUC1 was not downregulated in contrast to the expression of CAIX, a prognostic marker in gastric cancer. In conclusion, the HSP90 inhibitor ganetespib synergizes with platinum anticancer drugs and modulates intracellular signal transduction in direction of a less proliferative and aggressive phenotype.

Small cell lung cancer (SCLC) is disseminated in the majority of patients at first presentation and, thus, treated with chemoradiotherapy. Despite initial high response rates, chemoresistance appears rapidly and results in a dismal prognosis. However, patients with limited cancer may exhibit better disease control upon surgical treatment. Correct staging is highly critical in the selection of those patients which are likely to benefit from surgery. Studies of the inclusion of surgery in the multimodal treatment of SCLC vary widely in number of patients, selection, treatment and follow-up. Nevertheless surgical therapy for confined SCLCs achieves favorable long-term survival compared to chemoradiotherapy, depending on a precise assessment of the degree of tumor dissemination. Recently, extremely high counts of circulating tumor cells (CTCs) were reported in patients with SCLC compared to other malignancies. In several studies the number of CTCs was found to constitute a prognostic parameter and a marker of response to therapy. Therefore, the assessment of CTCs as so-called "Liquid Biopsy" seems to constitute a more precise method to detect tumor dissemination earlier when compared to clinical staging. In conclusion, in the era of precision oncology enumeration and identification of CTCs of SCLC patients have the potential to help in the selection of patients most suitable for tumor surgery.

Small cell lung cancer (SCLC) is distinguished by aggressive growth, early dissemination and a poor prognosis at advanced stage. The remarkably high count of circulating tumor cells (CTCs) of SCLC allowed for the establishment of permanent CTC cultures at our institution for the first time. CTCs are assumed to have characteristics of cancer stem cells (CSCs) and an epithelial-mesenchymal transition (EMT) phenotype, but extravasation of tumors at distal sites is marked by epithelial features. Two SCLC CTC cell lines, namely BHGc7 and BHGc10, as well as SCLC cell lines derived from primary tumors and metastases were analyzed for the expression of pluripotent stem cell markers and growth factors. Expression of E-cadherin and β-Catenin were determined by flow cytometry. Stem cell-associated markers SOX17, α-fetoprotein, OCT-3/4, KDR, Otx2, GATA-4, Nanog, HCG, TP63 and Goosecoid were not expressed in the 2 CTC lines. In contrast, high expression was found for HNF-3β/FOXA2, SOX2, PDX-1/IPF1 and E-cadherin. E-cadherin expression was restricted to the 2 CTCs and 2 cell lines derived from pleural effusion (SCLC26A) and bone metastases (NCI-H526), respectively. Thus, these SCLC CTCs established from extended disease SCLC patients lack expression of stem cell markers which suppress the epithelial phenotype. Instead they express high levels of E-cadherin consistent with a mesenchymal-epithelial transition (MET or EMrT) and form large tumorospheres possibly in response to the selection pressure of first-line chemotherapy. HNF-3β/FOXA2 and PDX-1/IPF1 expression seem to be related to growth factor dependence on insulin/IGF-1 receptors and IGF-binding proteins.

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.

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.