Barbara Rath

Small cell lung cancer: Recruitment of macrophages by circulating tumor cells

ABSTRACT Tumor-associated macrophages (TAMs) play an important role in tumor progression, suppression of antitumor immunity and dissemination. Blood monocytes infiltrate the tumor region and are primed by local microenvironmental conditions to promote tumor growth and invasion. Although many of the interacting cytokines and factors are known for the tumor-macrophage interactions, the putative contribution of circulating tumor cells (CTCs) is not known so far. These specialized cells are characterized by increased mobility, ability to degrade the extracellular matrix (ECM) and to enter the blood stream and generate secondary lesions which is a leading cause of death for the majority of tumor patients. The first establishment of two permanent CTC lines, namely BHGc7 and 10, from blood samples of advanced stage small cell lung cancer (SCLC) patients allowed us to investigate the CTC-immune cell interaction. Cocultures of peripheral blood mononuclear cells (PBMNCs) with CTCs or addition of CTC-conditioned medium (CTC-CM) in vitro resulted in monocyte-macrophage differentiation and appearance of CD14+, CD163weak and CD68+ macrophages expressing markers of TAMs. Furthermore, we screened the supernatants of CTC-primed macrophages for presence of approximately 100 cytokines and compared the expression with those induced by the local metastatic SCLC26A cell line. Macrophages recruited by SCLC26A-CM showed expression of osteopontin (OPN), monocyte chemoattractant protein-1 (MCP-1), IL-8, chitinase3-like 1 (CHI3L1), platelet factor (Pf4), IL-1ra and matrix metalloproteinase-9 (MMP-9) among other minor cytokines/chemokines. In contrast, BHGc7-CM induced marked overexpression of complement factor D (CFD)/adipsin and vitamin D-BP (VDBP), as well as increased secretion of OPN, lipocalin-2 (LCN2), CHI3L1, uPAR, MIP-1 and GDF-15/MIC-1. BHGc10, derived independently from relapsed SCLC, revealed an almost identical pattern with added expression of ENA-78/CXCL5. CMs of the non-tumor HEK293 cell line revealed no induction of macrophages, whereas incubation of PBMNCs with recombinant CHI3L1 gave positive results. Thus, the specific contributions of CTCs in SCLC affect CFD/adipsin, possibly involved in immunity/cachexia, VDBP which gives rise to group-specific component protein-derived macrophage-activating factor (GcMAF), GDF-15/MIC-1 which enhances the malignant phenotype of tumor cells and ENA-78/CXCL5 which attracts angiogenic neutrophils. In conclusion, CTCs are competent to specifically manipulate TAMs to increase invasiveness, angiogenesis, immunosuppression and possibly lipid catabolism.

Pharmacokinetics of crizotinib in NSCLC patients

Introduction: For a subpopulation of NSCLC patients genetic rearrangement of the anaplastic lymphoma kinase (ALK) was found as driver mutation, which can be targeted by the selective inhibitor crizotinib. Areas covered: This article presents an overview of the clinical studies that provided the characterization of the pharmacokinetic parameters for the administration of crizotinib to cancer patients and the factors influencing the clinical profiles of this drug. Expert opinion: Crizotinib is administered orally as a capsule and clinical studies indicated 250 mg crizotinib BID continuously as the maximal tolerated dose in cancer patients. Bioavailability is ∼ 40% and pharmacokinetic parameters are influenced by food only to a minor degree. This dose of the drug corresponds to a significant inhibition of the mutated ALK, retards tumor growth and achieves clinical responses in the majority of patients. Crizotinib lactam is the single metabolite with minor inhibitory activity for the ALK fusion protein. Metabolization is executed mainly by CYP3A4/5 and is modulated by other drugs interacting with this cytochrome oxidase. Despite the one-fits-all approach in administration of crizotinib at a fixed dose the pharmacokinetic parameters indicate a stable steady state upon continuous administration, which achieves sufficient inhibition of the ALK drug target.

Chitinase-3-like-1/YKL-40 as marker of circulating tumor cells.

Ex vivo expansion of circulating tumor cells (CTCs) of small cell lung cancer (SCLC) patients enabled systematic screening of secreted cytokines. Permanent CTC cultures of different patients shared secretion of chitinase-3-like-1 (CHI3L1)/YKL-40, known to be upregulated in a range of tumor entities and to be associated with increased metastasis and decreased survival. This protein lacks enzymatic activity and its mechanism of promoting tumor dissemination has not been resolved. Results from SCLC CTC cultures suggest CHI3L1 as marker and important effector of tumor cell dissemination in the peripheral blood. Furthermore, this protein may link chronic inflammation of the lung, chronic obstructive pulmonary disease (COPD) and lung cancer.

Synergism of cyclin-dependent kinase inhibitors with camptothecin derivatives in small cell lung cancer cell lines.

Advanced small cell lung cancer (SCLC) has a dismal prognosis. Modulation of the camptothecin topotecan, approved for second-line therapy, may improve response. Our recent finding of synergistic enhancement of the cytotoxic activity of camptothecin (CPT) by cyclin-dependent kinase 4 inhibitors is extended here to a panel of camptothecin analogs comprising 10-hydroxy-CPT (HOCPT), topotecan (TPT; 9-[(dimethylamino)-methyl]-10-hydroxy-CPT), 9-amino-CPT (9AC), 9-nitrocamptothecin (rubitecan), SN38 (7-ethyl-10-hydroxycamptothecin) and 10-hydroxy-9-nitrocamptothecin (CPT109) in combination with PD0332991, CDK4I, roscovitine and olomoucine. SCLC cell lines employed are chemoresistant NCI-H417 and DMS153 and the chemosensitive SCLC26A line established at our institution. The CPT analogs exhibiting highest cytotoxicity towards the three SCLC lines tested were SN38 and 9AC, followed by rubitecan, HOCPT, TPT and CPT109. NCI-H417 and DMS153 revealed an approximately 25-fold and 7-fold higher resistance compared to the chemosensitive SCLC26A cell line. Whereas the CDK4/6 inhibitor PD0332991 proved less effective to chemosensitize SCLC cells to CPT analogs, the CDK inhibitors CDK4I, roscovitine and olomoucine gave comparable chemosensitization effects in combination with 9AC, SN38, rubitecan and to a lesser extent with TPT and CPT109, not directly related with topoisomerase mRNA expression. In conclusion, small chemical modifications of the parent CPT structure result in differing cytotoxicities and chemomodulatory effects in combination with CDKIs of the resulting analogs.

Avelumab: combining immune checkpoint inhibition and antibody-dependent cytotoxicity

ABSTRACT Introduction: Immune checkpoint inhibition holds great promise for selected tumors. The human monoclonal antibody (mAB) avelumab is directed to programmed death ligand-1 (PD-L1) and is supposed to inhibit the immunosuppressive PD-L1/PD-1 interaction and, furthermore, effect antibody-dependent cytotoxicity (ADCC) lysis of tumor cells. Areas covered: This article presents an overview of the current means to activate the antitumor immune defense by targeting PD-1 or PD-L1 with mABs and their possible role in ADCC-mediated tumor cell elimination. Expert opinion: Avelumab contains a Fc region which can bind cognate receptors on immune effector cells and induce ADCC-mediated tumor cell lysis, in contrast to other mABs directed to PD-1/PD-L1 which lack the ability to trigger ADCC due to belonging to the IgG4 subclass or possessing a mutated Fc region. Preclinical and clinical data indicate that avelumab can be safely administered to cancer patients with a toxicity profile comparable to other mABs and without lysis of PD-L1-positive activated immune cells. This antibody yielded durable responses in a phase II trial in advanced Merkel cell carcinoma patients. Tumor cell lysis by avelumab prevents cells from resorting to alternative checkpoints as shown by targeting PD-1 and the upregulation of TIM-3.

A short update on cancer chemoresistance

SummaryChemotherapeutic interventions in cancer patients are limited by the appearance of chemoresistance. For instance, advanced lung and ovarian cancer patients relapse invariably after few cycles of platinum-based chemotherapy. Disseminated tumors are characterized by genetic instability/heterogeneity, thus containing or generating a repertoire of resistant subpopulations. At the cellular level, altered drug uptake, efflux, and metabolization, as well as modifications of drug targets, increased repair, and decreased cell death complement the limited perfusion and adverse hypoxic/acidic extracellular conditions at the tumor level in retaining cancer cell viability. Similarly, targeted therapy is rendered ineffective by mutations of the specific target protein within a few months or years of administration. Assessment of the expression profiles of resistant tumor cells revealed extensive changes in numerous pathways affecting hundreds of genes. Therefore, reversal of drug resistance will require individual profiles of drug resistance mediators and the combination of several specific drugs, targeting critical components to provide new therapeutic options.ZusammenfassungDie Chemotherapie wird in Krebspatienten durch das Auftreten von Chemoresistenz begrenzt. Zum Beispiel rezidivieren Lungen- und Ovarialkarzinompatienten schon nach wenigen Zyklen der platinbasierten Chemotherapie. Disseminierte Tumoren weisen genetische Instabilität/Heterogenität auf und enthalten oder generieren resistente Subpopulationen. Auf der Zellebene komplementieren veränderte Wirkstoffaufnahme, -efflux und –metabolisierung, Modifikationen der Zielproteine, gesteigerte Reparaturprozesse und verminderter Zelltod die herabgesetzte Perfusion und die ungünstigen hypoxischen/azidotischen Bedingungen auf Tumorebene, die die Viabilität der Tumorzellen erhalten. In ähnlicher Weise wird bei der zielgerichteten Chemotherapie die Wirksamkeit durch Mutationen des Zielproteins innerhalb weniger Monate oder Jahre herabgesetzt. Eine Erfassung der Expressionsprofile resistenter Tumorzellen zeigen Änderungen in zahlreichen Stoffwechselwegen die hunderte Gene betreffen. Folglich verlangt die erfolgreiche Reversion der Resistenz die Erfassung individueller Profile der verantwortlichen Mediatoren und den Einsatz einer Kombination von Wirkstoffen gegen kritische Zielproteine.

Second-line therapy for small cell lung cancer: exploring the potential role of circulating tumor cells

BACKGROUND Small cell lung cancer (SCLC) is an aggressive and invasive variant of lung tumors. SCLC, especially in advanced stages, is distinguished by extremely high numbers of circulating tumor cells (CTCs) in comparison to other malignancies. CTCs are operative in tumor spread and are currently enumerated to assess prognosis and response to cytotoxic therapy. Chemosensitivity of SCLC CTCs compared to primary tumors and metastases is not known. METHODS Establishment of two SCLC CTC cell lines, namely BHGc7 and BHGc10, allowed the in vitro characterization of their chemosensitivity to the second-line chemotherapeutics topotecan and epirubicin in comparison to a range of SCLC cell lines. RESULTS The SCLC CTC cell lines exhibited an approximately 7- and 12-fold increased chemosensitivity to epirubicin compared to topotecan, respectively, in in vitro cytotoxicity assays. In comparison to a panel of six SCLC cell lines, the two CTC lines showed a significantly higher chemosensitivity to epirubicin (range, 3- to 16-fold) and topotecan (range, 2.2- to 14.4-fold), respectively. CONCLUSIONS CTC cell lines derived from SCLC patients with recurrent disease exhibit high chemosensitivity to epirubicin vs. topotecan and show considerable more cytotoxicity in response to both compounds in comparison to a panel of SCLC cell lines. Thus, a decrease in the number of CTCs in response to second-line chemotherapy in SCLC patients may overestimate the effect on resident SCLC lesions and metastases.

Detection of circulating tumor cells in non-small cell lung cancer

Lung cancer represents the second most common type of cancer in both men and women in the Western world and is a leading cause of mortality (1). Approximately 80% of all lung cancers are classed as NSCLC, 15% are small cell lung cancer (SCLC) and other histological variants account for about 5% (2). Although NSCLC comprises different histological types, prognosis and therapy for the approximately 40% adenocarcinomas, 25% to 30% squamous cell carcinomas (SCCs) and 10% to 15% large cell carcinomas (LCC) is often comparable. For patients with early-stage disease, surgery followed by adjuvant chemotherapy is the optimal treatment (3). However, over 50% of patients are initially diagnosed with advanced or metastatic disease with worse outcomes. For patients with locally advanced disease, the standard approach is chemoradiotherapy since it offers a small but statistically significant prolongation in survival. For patients with metastatic disease, chemotherapy represents the mainstay of treatment resulting in a median survival of approximately 10 months. The 5-year survival rate for metastatic disease is a low as 3%, whereas the 5-year survival rate for all stages is approximately 15%. Therefore, early detection of tumor dissemination and further improvement in diagnostics and treatments are required.

Small cell lung cancer: model of circulating tumor cell tumorospheres in chemoresistance

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.

Smoking, inflammation and small cell lung cancer: recent developments

SummarySmall cell lung cancer (SCLC) accounts for 15 % of all lung tumors and represents an invasive neuroendocrine malignancy with poor survival rates. This cancer is highly prevalent in smokers and characterized by inactivation of p53 and retinoblastoma. First in vitro expansion of circulating tumor cells (CTCs) of SCLC patients allowed for investigation of the cell biology of tumor dissemination. In the suggested CTC SCLC model, the primary tumor attracts and educates tumor-promoting and immunosuppressive macrophages which in turn arm CTCs to spread and generate distal lesions. Preexisting inflammatory processes associated with chronic obstructive pulmonary disease (COPD) seem to potentiate the subsequent activity of tumor-associated macrophages (TAM). Activation of signal transducer and activator of transcription 3 (STAT3) and expression of chitinase-3-like 1/YKL-40 in SCLC CTCs seems to be associated with drug resistance. In conclusion, inflammation-associated generation of invasive and chemoresistant CTCs most likely explains the characteristic features of SCLC, namely early dissemination and rapid failure of chemotherapy.ZusammenfassungDas kleinzelliges Lungenkarzinom (SCLC) ist für 15 % der Lungenkarzinome verantwortlich und stellt eine aggressiv wachsende und invasive neuroendokrine maligne Erkrankung dar, die mit niedrigen Überlebensarten verbunden ist. Dieses Karzinom weist eine hohe Inzidenz bei Rauchern auf und ist vor allem durch die Inaktivierung von p53 und Retinoblastom-Gen charakterisiert. Die erstmalige in vitro Kultur von zirkulierenden Tumorzellen (CTCs) von SCLC Patienten ermöglichte die Untersuchung der Zellbiologie der Tumordissemination. Bei dem vorgeschlagenen Modell der SCLC CTCs rekrutiert der Tumor Makrophagen mit einem wachstumsfördernden und immunsuppressiven Phänotyp, der wiederum CTCs hervorbringt, die invasiv Fernmetastasen setzen. Bereits bestehende Entzündungsprozesse durch chronische obstruktive pulmonäre Erkrankung (COPD) scheinen die nachfolgende Aktivität der tumorassoziierten Makrophagen (TAM) zu potenzieren. Die Aktivierung von STAT3 und Chitinase-3-like-1/YKL-40 in den CTCs führt wahrscheinlich zur Chemoresistenz. Zusammenfassend führt die mit Entzündungen verbundene Entwicklung invasiver und chemoresistenter CTCs zu den typischen Charakteristika des kleinzelligen Lungenkarzinoms, nämlich früher Metastasierung und frühem Versagen der Chemotherapie und damit verweist dieses Modell des SCLC auf neue therapeutische Angriffspunkte.