Tobias Pukrop

Macrophages Induce Invasiveness of Epithelial Cancer Cells Via NF-κB and JNK

Tumor-associated macrophages may influence tumor progression, angiogenesis and invasion. To investigate mechanisms by which macrophages interact with tumor cells, we developed an in vitro coculture model. Previously we reported that coculture enhanced invasiveness of the tumor cells in a TNF-α- and matrix metalloprotease-dependent manner. In this report, we studied intracellular signaling pathways and induction of inflammatory genes in malignant cells under the influence of macrophage coculture. We report that coculture of macrophages with ovarian or breast cancer cell lines led to TNF-α-dependent activation of JNK and NF-κB pathways in tumor cells, but not in benign immortalized epithelial cells. Tumor cells with increased JNK and NF-κB activity exhibited enhanced invasiveness. Inhibition of the NF-κB pathway by TNF-α neutralizing Abs, an NF-κB inhibitor, RNAi to RelA, or overexpression of IκB inhibited tumor cell invasiveness. Blockade of JNK also significantly reduced invasiveness, but blockade of p38 MAPK or p42 MAPK had no effect. Cocultured tumor cells were screened for the expression of 22 genes associated with inflammation and invasion that also contained an AP-1 and NF-κB binding site. EMMPRIN and MIF were up-regulated in cocultured tumor cells in a JNK- and NF-κB-dependent manner. Knocking down either MIF or EMMPRIN by RNAi in the tumor cells significantly reduced tumor cell invasiveness and matrix metalloprotease activity in the coculture supernatant. We conclude that TNF-α, via NF-κB, and JNK induces MIF and EMMPRIN in macrophage to tumor cell cocultures and this leads to increased invasive capacity of the tumor cells.

Microglia promote colonization of brain tissue by breast cancer cells in a Wnt-dependent way.

Although there is increasing evidence that blood‐derived macrophages support tumor progression, it is still unclear whether specialized resident macrophages, such as brain microglia, also play a prominent role in metastasis formation. Here, we show that microglia enhance invasion and colonization of brain tissue by breast cancer cells, serving both as active transporters and guiding rails. This is antagonized by inactivation of microglia as well as by the Wnt inhibitor Dickkopf‐2. Proinvasive microglia demonstrate altered morphology, but neither upregulation of M2‐like cytokines nor differential gene expression. Bacterial lipopolysacharide shifts tumor‐educated microglia into a classical M1 phenotype, reduces their proinvasive function, and unmasks inflammatory and Wnt signaling as the most strongly regulated pathways. Histological findings in human brain metastases underline the significance of these results. In conclusion, microglia are critical for the successful colonization of the brain by epithelial cancer cells, suggesting inhibition of proinvasive microglia as a promising antimetastatic strategy.

Chemoimmunotherapy with methotrexate, cytarabine, thiotepa, and rituximab (MATRix regimen) in patients with primary CNS lymphoma: results of the first randomisation of the International Extranodal Lymphoma Study Group-32 (IELSG32) phase 2 trial

BACKGROUND Standard treatment for patients with primary CNS lymphoma remains to be defined. Active therapies are often associated with increased risk of haematological or neurological toxicity. In this trial, we addressed the tolerability and efficacy of adding rituximab with or without thiotepa to methotrexate-cytarabine combination therapy (the MATRix regimen), followed by a second randomisation comparing consolidation with whole-brain radiotherapy or autologous stem cell transplantation in patients with primary CNS lymphoma. We report the results of the first randomisation in this Article. METHODS For the international randomised phase 2 International Extranodal Lymphoma Study Group-32 (IELSG32) trial, HIV-negative patients (aged 18-70 years) with newly diagnosed primary CNS lymphoma and measurable disease were enrolled from 53 cancer centres in five European countries (Denmark, Germany, Italy, Switzerland, and the UK) and randomly assigned (1:1:1) to receive four courses of methotrexate 3·5 g/m(2) on day 1 plus cytarabine 2 g/m(2) twice daily on days 2 and 3 (group A); or the same combination plus two doses of rituximab 375 mg/m(2) on days -5 and 0 (group B); or the same methotrexate-cytarabine-rituximab combination plus thiotepa 30 mg/m(2) on day 4 (group C), with the three groups repeating treatment every 3 weeks. Patients with responsive or stable disease after the first stage were then randomly allocated between whole-brain radiotherapy and autologous stem cell transplantation. A permuted blocks randomised design (block size four) was used for both randomisations, and a computer-generated randomisation list was used within each stratum to preserve allocation concealment. Randomisation was stratified by IELSG risk score (low vs intermediate vs high). No masking after assignment to intervention was used. The primary endpoint of the first randomisation was the complete remission rate, analysed by modified intention to treat. This study is registered with ClinicalTrials.gov, number NCT01011920. FINDINGS Between Feb 19, 2010, and Aug 27, 2014, 227 eligible patients were recruited. 219 of these 227 enrolled patients were assessable. At median follow-up of 30 months (IQR 22-38), patients treated with rituximab and thiotepa had a complete remission rate of 49% (95% CI 38-60), compared with 23% (14-31) of those treated with methotrexate-cytarabine alone (hazard ratio 0·46, 95% CI 0·28-0·74) and 30% (21-42) of those treated with methotrexate-cytarabine plus rituximab (0·61, 0·40-0·94). Grade 4 haematological toxicity was more frequent in patients treated with methotrexate-cytarabine plus rituximab and thiotepa, but infective complications were similar in the three groups. The most common grade 3-4 adverse events in all three groups were neutropenia, thrombocytopenia, anaemia, and febrile neutropenia or infections. 13 (6%) patients died of toxicity. INTERPRETATION With the limitations of a randomised phase 2 study design, the IELSG32 trial provides a high level of evidence supporting the use of MATRix combination as the new standard chemoimmunotherapy for patients aged up to 70 years with newly diagnosed primary CNS lymphoma and as the control group for future randomised trials. FUNDING Associazione Italiana del Farmaco, Cancer Research UK, Oncosuisse, and Swiss National Foundation.

β-catenin-independent WNT signaling in basal-like breast cancer and brain metastasis.

A role of WNT signaling for primary breast cancers of the basal-like subtype and as a predictor of brain metastasis has been described. However, a responsible WNT ligand has not been identified. To further clarify this question, we comparatively investigated 22 human breast cancer brain metastases as well as the highly invasive human breast cancer cell line MDA-MB-231 and the weakly motile MCF-7 as models for the basal-like and the luminal A subtype. WNT5A and B were found overexpressed in MDA-MB-231 cells as compared with MCF-7. This corresponded to reduction of MDA-MB-231 invasiveness by WNT inhibitors, whereas MCF-7 invasion was enhanced by recombinant WNT5B and abolished by WNT and Jun-N-terminal kinase antagonists. Expression and subcellular distribution of β-catenin remained uninfluenced. Consistently, β-catenin was not localized in the nuclei of brain metastases while there was strong nuclear c-Jun staining. Similar to MDA-MB-231, metastases showed expression of WNT5A/B and the alternative WNT receptors ROR1 and 2. These findings were validated using external gene expression datasets (Gene Expression Omnibus) of different breast cancer subtypes and brain metastases. Hierarchical cluster analysis yielded a close relation between basal-like cancers and brain metastases. Gene set enrichment analyses confirmed WNT pathway enrichment not only in basal-like primaries but also in cerebral metastases of all subtypes. In conclusion, WNT signaling seems highly relevant for basal-like and other subtypes of breast cancers metastasizing into the brain. β-catenin-independent WNT signaling, presumably via ROR1-2, plays a major role in this context.

Toll-like receptor activation reveals developmental reorganization and unmasks responder subsets of microglia.

The sentinel and immune functions of microglia require rapid and appropriate reactions to infection and damage. Their Toll‐like receptors (TLRs) sense both as threats. However, whether activated microglia mount uniform responses or whether subsets conduct selective tasks is unknown. We demonstrate that murine microglia reorganize their responses to TLR activations postnatally and that this process comes with a maturation of TLR4‐organized functions. Although induction of MHCI for antigen presentation remains as a pan‐populational feature, synthesis of TNFα becomes restricted to a subset, even within adult central nervous system regions. Response heterogeneity is evident ex vivo, in situ, and in vivo, but is not limited to TNFα production or to TLR‐triggered functions. Also, clearance activities for myelin under physiological and pathophysiological conditions, IFNγ‐enforced upregulation of MHCII, or challenged inductions of other proinflammatory factors reveal dissimilar microglial contributions. Notably, response heterogeneity is also confirmed in human brain tissue. Our findings suggest that microglia divide by constitutive and inducible capacities. Privileged production of inflammatory mediators assigns a master control to subsets. Sequestration of clearance of endogenous material versus antigen presentation in exclusive compartments can separate potentially interfering functions. Finally, subsets rather than a uniform population of microglia may assemble the reactive phenotypes in responses during infection, injury, and rebuilding, warranting consideration in experimental manipulation and therapeutic strategies.

The complex pathways of Wnt 5a in cancer progression

In contrast to the transforming members of the Wnt family, shown to be upregulated in many cancers, the role of Wnt 5a is still controversial. While it has been attributed a tumour suppressor function in some malignancies, there is increasing evidence of promigratory and proinvasive effects in others, mediated predominantly through the planar cell polarity pathway and activation of protein kinase C. Obviously, the outcome of an individual Wnt 5a signal is dependent on a multitude of variables, ranging from availability of receptors, downstream effectors, and inhibitors to external influences coming from the tumour microenvironment and the extracellular matrix.

Silencing of the Wnt transcription factor TCF4 sensitizes colorectal cancer cells to (chemo-) radiotherapy.

A considerable percentage of rectal cancers are resistant to standard preoperative chemoradiotherapy. Because patients with a priori-resistant tumors do not benefit from multimodal treatment, understanding and overcoming this resistance remains of utmost clinical importance. We recently reported overexpression of the Wnt transcription factor TCF4, also known as TCF7L2, in rectal cancers that were resistant to 5-fluorouracil-based chemoradiotherapy. Because Wnt signaling has not been associated with treatment response, we aimed to investigate whether TCF4 mediates chemoradioresistance. RNA interference-mediated silencing of TCF4 was employed in three colorectal cancer (CRC) cell lines, and sensitivity to (chemo-) radiotherapy was assessed using a standard colony formation assay. Silencing of TCF4 caused a significant sensitization of CRC cells to clinically relevant doses of X-rays. This effect was restricted to tumor cells with high T cell factor (TCF) reporter activity, presumably in a β-catenin-independent manner. Radiosensitization was the consequence of (i) a transcriptional deregulation of Wnt/TCF4 target genes, (ii) a silencing-induced G(2)/M phase arrest, (iii) an impaired ability to adequately halt cell cycle progression after radiation and (iv) a compromised DNA double strand break repair as assessed by γH2AX staining. Taken together, our results indicate a novel mechanism through which the Wnt transcription factor TCF4 mediates chemoradioresistance. Moreover, they suggest that TCF4 is a promising molecular target to sensitize resistant tumor cells to (chemo-) radiotherapy.

Impact of teaching and assessment format on electrocardiogram interpretation skills

Medical Education 2010: 44: 731–740

Tumor-derived microvesicles mediate human breast cancer invasion through differentially glycosylated EMMPRIN

Tumor cells secrete not only a variety of soluble factors, but also extracellular vesicles that are known to support the establishment of a favorable tumor niche by influencing the surrounding stroma cells. Here we show that tumor-derived microvesicles (T-MV) also directly influence the tumor cells by enhancing their invasion in a both autologous and heterologous manner. Neither the respective vesicle-free supernatant nor MV from benign mammary cells mediate invasion. Uptake of T-MV is essential for the proinvasive effect. We further identify the highly glycosylated form of the extracellular matrix metalloproteinase inducer (EMMPRIN) as a marker for proinvasive MV. EMMPRIN is also present at high levels on MV from metastatic breast cancer patients in vivo. Anti-EMMPRIN strategies, such as MV deglycosylation, gene knockdown, and specific blocking peptides, inhibit MV-induced invasion. Interestingly, the effect of EMMPRIN-bearing MV is not mediated by matrix metalloproteinases but by activation of the p38/MAPK signaling pathway in the tumor cells. In conclusion, T-MV stimulate cancer cell invasion via a direct feedback mechanism dependent on highly glycosylated EMMPRIN.

Carcinoma cells misuse the host tissue damage response to invade the brain

The metastatic colonization of the brain by carcinoma cells is still barely understood, in particular when considering interactions with the host tissue. The colonization comes with a substantial destruction of the surrounding host tissue. This leads to activation of damage responses by resident innate immune cells to protect, repair, and organize the wound healing, but may distract from tumoricidal actions. We recently demonstrated that microglia, innate immune cells of the CNS, assist carcinoma cell invasion. Here we report that this is a fatal side effect of a physiological damage response of the brain tissue. In a brain slice coculture model, contact with both benign and malignant epithelial cells induced a response by microglia and astrocytes comparable to that seen at the interface of human cerebral metastases. While the glial damage response intended to protect the brain from intrusion of benign epithelial cells by inducing apoptosis, it proved ineffective against various malignant cell types. They did not undergo apoptosis and actually exploited the local tissue reaction to invade instead. Gene expression and functional analyses revealed that the C‐X‐C chemokine receptor type 4 (CXCR4) and WNT signaling were involved in this process. Furthermore, CXCR4‐regulated microglia were recruited to sites of brain injury in a zebrafish model and CXCR4 was expressed in human stroke patients, suggesting a conserved role in damage responses to various types of brain injuries. Together, our findings point to a detrimental misuse of the glial damage response program by carcinoma cells resistant to glia‐induced apoptosis. GLIA 2013;61:1331–1346