TheraVision: Engineering platform technology for the development of oncolytic viruses based on herpes simplex virus type 1.

Viruses are able to efficiently penetrate cells, multiply, and eventually kill infected cells, release tumor antigens, and activate the immune system. Therefore, viruses are highly attractive novel agents for cancer therapy. Clinical trials with first generations of oncolytic viruses (OVs) are very promising but show significant need for optimization. The aim of TheraVision was to establish a broadly applicable engineering platform technology for combinatorial oncolytic virus and immunotherapy. Through genetic engineering, an attenuated herpes simplex virus type 1 (HSV1) was generated that showed increased safety compared to the wild-type strain. To demonstrate the modularity and the facilitated generation of new OVs, two transgenes encoding retargeting as well as immunomodulating single-chain variable fragments (scFvs) were integrated into the platform vector. The resulting virus selectively infected epidermal growth factor receptor (EGFR)-expressing cells and produced a functional immune checkpoint inhibitor against programmed cell death protein 1 (PD-1). Thus, both viral-mediated oncolysis and immune-cell-mediated therapy were combined into a single viral vector. Safety and functionality of the armed OVs have been shown in novel preclinical models ranging from patient-derived organoids and tissue-engineered human in vitro 3D tumor models to complex humanized mouse models. Consequently, a novel and proprietary engineering platform vector based on HSV1 is available for the facilitated preclinical development of oncolytic virotherapy.

Ex vivo expansion of lung cancer-derived disseminated cancer cells from lymph nodes identifies cells associated with metastatic progression.

The cellular basis of the apparent aggressiveness in lung cancer is poorly understood but likely associated with functional or molecular features of disseminated cancer cells (DCCs). DCCs from epithelial cancers are mostly detected by antibodies directed against histogenetic markers such as cytokeratin or EpCAM. It has been argued that marker-negative metastatic founder cells might escape detection. We therefore used ex vivo sphere formation for functional detection of candidate metastasis founders. We generated cell suspensions from 199 LN samples of 131 lung cancer patients and placed them into non-adherent cell culture. Sphere formation was associated with detection of DCCs using EpCAM immunocytology and with significantly poorer prognosis. The prognostic impact of sphere formation was strongly associated with high numbers of EpCAM-positive DCCs and aberrant genotypes of expanded spheres. We also noted sphere formation in patients with no evidence of lymphatic spread, however such spheres showed infrequent expression of signature genes associated with spheres from EpCAM-positive samples and displayed neither typical lung cancer mutations (KRAS, TP53, ERBB1) nor copy number variations, but might be linked to disease progression >5 years post curative surgery. We conclude that EpCAM identifies relevant disease-driving DCCs, that such cells can be expanded for model generation and that further research is needed to clarify the functional and prognostic role of rare EpCAM-negative sphere forming cells.

Single-cell microRNA sequencing method comparison and application to cell lines and circulating lung tumor cells.

Molecular single cell analyses provide insights into physiological and pathological processes. Here, in a stepwise approach, we first evaluate 19 protocols for single cell small RNA sequencing on MCF7 cells spiked with 1 pg of 1,006 miRNAs. Second, we analyze MCF7 single cell equivalents of the eight best protocols. Third, we sequence single cells from eight different cell lines and 67 circulating tumor cells (CTCs) from seven SCLC patients. Altogether, we analyze 244 different samples. We observe high reproducibility within protocols and reads covered a broad spectrum of RNAs. For the 67 CTCs, we detect a median of 68 miRNAs, with 10 miRNAs being expressed in 90% of tested cells. Enrichment analysis suggested the lung as the most likely organ of origin and enrichment of cancer-related categories. Even the identification of non-annotated candidate miRNAs was feasible, underlining the potential of single cell small RNA sequencing.

Interleukin-6 trans-signaling is a candidate mechanism to drive progression of human DCCs during clinical latency.

Although thousands of breast cancer cells disseminate and home to bone marrow until primary surgery, usually less than a handful will succeed in establishing manifest metastases months to years later. To identify signals that support survival or outgrowth in patients, we profile rare bone marrow-derived disseminated cancer cells (DCCs) long before manifestation of metastasis and identify IL6/PI3K-signaling as candidate pathway for DCC activation. Surprisingly, and similar to mammary epithelial cells, DCCs lack membranous IL6 receptor expression and mechanistic dissection reveals IL6 trans-signaling to regulate a stem-like state of mammary epithelial cells via gp130. Responsiveness to IL6 trans-signals is found to be niche-dependent as bone marrow stromal and endosteal cells down-regulate gp130 in premalignant mammary epithelial cells as opposed to vascular niche cells. PIK3CA activation renders cells independent from IL6 trans-signaling. Consistent with a bottleneck function of microenvironmental DCC control, we find PIK3CA mutations highly associated with late-stage metastatic cells while being extremely rare in early DCCs. Our data suggest that the initial steps of metastasis formation are often not cancer cell-autonomous, but also depend on microenvironmental signals.

Genetic alterations driving metastatic colony formation are acquired outside of the primary tumour in melanoma.

Mouse models indicate that metastatic dissemination occurs extremely early; however, the timing in human cancers is unknown. We therefore determined the time point of metastatic seeding relative to tumour thickness and genomic alterations in melanoma. Here, we find that lymphatic dissemination occurs shortly after dermal invasion of the primary lesion at a median thickness of ~0.5 mm and that typical driver changes, including BRAF mutation and gained or lost regions comprising genes like MET or CDKNA2, are acquired within the lymph node at the time of colony formation. These changes define a colonisation signature that was linked to xenograft formation in immunodeficient mice and death from melanoma. Thus, melanoma cells leave primary tumours early and evolve at different sites in parallel. We propose a model of metastatic melanoma dormancy, evolution and colonisation that will inform direct monitoring of adjuvant therapy targets.

Early dissemination seeds metastasis in breast cancer.

Accumulating data suggest that metastatic dissemination often occurs early during tumour formation, but the mechanisms of early metastatic spread have not yet been addressed. Here, by studying metastasis in a HER2-driven mouse breast cancer model, we show that progesterone-induced signalling triggers migration of cancer cells from early lesions shortly after HER2 activation, but promotes proliferation in advanced primary tumour cells. The switch from migration to proliferation was regulated by increased HER2 expression and tumour-cell density involving microRNA-mediated progesterone receptor downregulation, and was reversible. Cells from early, low-density lesions displayed more stemness features, migrated more and founded more metastases than cells from dense, advanced tumours. Notably, we found that at least 80% of metastases were derived from early disseminated cancer cells. Karyotypic and phenotypic analysis of human disseminated cancer cells and primary tumours corroborated the relevance of these findings for human metastatic dissemination.

Immune humanization of immunodeficient mice using diagnostic bone marrow aspirates from carcinoma patients.

Tumor xenografts in immunodeficient mice, while routinely used in cancer research, preclude studying interactions of immune and cancer cells or, if humanized by allogeneic immune cells, are of limited use for tumor-immunological questions. Here, we explore a novel way to generate cancer models with an autologous humanized immune system. We demonstrate that hematopoietic stem and progenitor cells (HSPCs) from bone marrow aspirates of non-metastasized carcinoma patients, which are taken at specialized centers for diagnostic purposes, can be used to generate a human immune system in NOD-scid IL2rγ(null) (NSG) and HLA-I expressing NSG mice (NSG-HLA-A2/HHD) comprising both, lymphoid and myeloid cell lineages. Using NSG-HLA-A2/HHD mice, we show that responsive and self-tolerant human T cells develop and human antigen presenting cells can activate human T cells. As critical factors we identified the low potential of bone marrow HSPCs to engraft, generally low HSPC numbers in patient-derived bone marrow samples, cryopreservation and routes of cell administration. We provide here an optimized protocol that uses a minimum number of HSPCs, preselects high-quality bone marrow samples defined by the number of initially isolated leukocytes and intra-femoral or intra-venous injection. In conclusion, the use of diagnostic bone marrow aspirates from non-metastasized carcinoma patients for the immunological humanization of immunodeficient mice is feasible and opens the chance for individualized analyses of anti-tumoral T cell responses.

A combination of hypoxia and lipopolysaccharide activates tristetraprolin to destabilize proinflammatory mRNAs such as tumor necrosis factor-alpha.

Inflammation is often accompanied by hypoxia because of the high oxygen consumption of invading bacteria and immune cells. During resolution of inflammation, the formation of inflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha), which is produced by macrophages, needs to be terminated. We show in RAW264.7 macrophages that TNF-alpha mRNA as well as intracellular and secreted TNF-alpha protein levels are reduced after prolonged incubations with lipopolysaccharide (LPS) under hypoxic conditions. The decrease in TNF-alpha was mediated by destabilization of TNF-alpha mRNA via a 3'-untranslated region-dependent mechanism. Specifically, the RNA-binding protein tristetraprolin (TTP) increased at mRNA and protein levels after 16-hour incubations with LPS under hypoxia. Interestingly, TTP accumulated in a dephosphorylated and active form, and this accumulation was attributable to reduced p38 mitogen-activated protein kinase activity under these conditions. Knockdown of TTP by small interfering RNA abolished destabilization of TNF-alpha mRNA. Prolonged incubations with LPS under hypoxia also reduced mRNA amounts and stability of other proinflammatory mediators such as macrophage inflammatory protein-2, interleukin-6, and granulocyte macrophage colony-stimulating factor. Therefore, we propose that hypoxia plays a key role during resolution of inflammation by activating posttranscriptional, TTP-dependent regulatory mechanisms.

Roles of hypoxia-inducible factor-1alpha (HIF-1alpha) versus HIF-2alpha in the survival of hepatocellular tumor spheroids.

UNLABELLED: Hypoxia-inducible factors (HIFs) provoke adaptation to hypoxic stress occurring in rapidly growing tumor tissues. Therefore, overexpression of HIF-1 or HIF-2 is a common feature in hepatocellular carcinoma but their specific function is still controversially discussed. To analyze HIF function in hypoxia-induced cell death we created a stable knockdown of HIF-1alpha and HIF-2alpha in HepG2 cells and generated tumor spheroids as an in vitro hepatocellular carcinoma model. Knockdown of HIF-1alpha enhanced expression of HIF-2alpha and vice versa. Unexpectedly, knockdown of HIF-1alpha or HIF-2alpha increased cell viability as well as spheroid size and decreased caspase-3 activity. Antiapoptotic Bcl-X(L) expression increased in both knockdown spheroids, whereas proapoptotic Bax was only reduced in HIF-1alpha-knockdown cells. Furthermore, an HIF-2alpha-knockdown significantly increased Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) expression in an HIF-1alpha-dependent manner. Concomitantly, electron microscopy revealed a substantial increase in autophagosomal structures in HIF-2alpha-knockdown spheroids and mito-/lysotracker costaining confirmed lysosomal activity of these autophagosomes. Blocking autophagosome maturation using 3-methyladenine restored cell death in HIF-2alpha-knockdown clones comparable to wildtype cells. CONCLUSION: An HIF-1alpha-knockdown increases HIF-2alpha expression and shifts the balance of Bcl-2 family members toward survival. The knockdown of HIF-2alpha raises autophagic activity and attenuates apoptosis by enhancing HIF-1alpha expression. Our data indicate that enhanced expression of one HIF-isoform causes a survival advantage in hepatocellular carcinoma development.

Knockout of HIF-1α in tumor-associated macrophages enhances M2 polarization and attenuates their pro-angiogenic responses.

Tumor-associated macrophages (TAMs) constitute major infiltrates of solid tumors and express a marker profile that characterizes alternatively activated macrophages (MФs). TAMs accumulate in hypoxic tumor regions, express high amounts of hypoxia-inducible factor-1 (HIF-1) and contribute to tumor angiogenesis and invasiveness. However, the precise role of HIF-1 on MФ infiltration and phenotype alterations remains poorly defined. Therefore, we cocultured wild type (wt) versus HIF-1α(-/-) MФs with tumor spheroids. Both, wt and HIF-1α(-/-) MФs, infiltrated hypoxic regions of tumor spheroids at equal rates and got alternatively activated. Interestingly, significantly higher amounts of HIF-1α(-/-) MФs expressed the TAM markers CD206 and stabilin-1 compared with wt phagocytes. Stimulation of infiltrated TAMs with lipopolysaccharide (LPS)/interferon-γ revealed a reduced expression of the pro-inflammatory markers interleukin (IL)-6, tumor necrosis factor-α and inducible nitric oxide synthase in HIF-1α(-/-) MФs. Furthermore, HIF-1α(-/-) MФs were less cytotoxic toward tumor cells. Although infiltration of MФs increased the invasive potential of tumor spheroids independently of HIF-1, the ability to stimulate differentiation of stem cells toward CD31-positive cells was triggered by wt but not by HIF-1α(-/-) MФs. Our data suggest that HIF-1α-deficient MФs develop a more prominent TAM marker profile accompanied by reduced cytotoxicity, whereas HIF-1 seems indispensable for the angiogenesis-promoting properties of TAMs.

Sphingosine kinase 2 deficient tumor xenografts show impaired growth and fail to polarize macrophages towards an anti-inflammatory phenotype.

A challenging task of the immune system is to fight cancer cells. However, a variety of human cancers educate immune cells to become tumor supportive. This is exemplified for tumor-associated macrophages (TAMs), which are polarized towards an anti-inflammatory and cancer promoting phenotype. Mechanistic explanations, how cancer cells influence the macrophage phenotype are urgently needed to address potential anti-cancer strategies along this line. One potential immune modulating compound, sphingosine-1-phosphate (S1P), was recently highlighted in both tumor growth and immune modulation. Using a xenograft model in nude mice, we demonstrate a supportive role of sphingosine kinase 2 (SphK2), one of the S1P-producing enzymes for tumor progression. The growth of SphK2-deficient MCF-7 breast tumor xenografts was markedly delayed when compared with controls. Infiltration of macrophages in SphK2-deficient and control tumors was comparable. However, TAMs from SphK2-deficient tumors displayed a pronounced anti-tumor phenotype, showing an increased expression of pro-inflammatory markers/mediators such as NO, TNF-alpha, IL-12 and MHCII and a low expression of anti-inflammatory IL-10 and CD206. These data suggest a role for S1P, generated by SphK2, in early tumor development by affecting macrophage polarization.

The supernatant of apoptotic cells causes transcriptional activation of hypoxia-inducible factor-1alpha in macrophages via sphingosine-1-phosphate and transforming growth factor-beta.

Macrophages infiltrating solid tumors exhibit a tumor-supporting phenotype and are critical for tumor development. Little is known which tumor-derived signal provokes this phenotype shift and how these signals are interpreted in macrophages to support tumor growth. We used the supernatant of apoptotic cells and noticed transcriptional, nuclear factor of activated T cells-dependent up-regulation of hypoxia-inducible factor (HIF)-1alpha mRNA, subsequent protein expression, and HIF-1 activity. Blocking calcineurin with cyclosporine A attenuated nuclear factor of activated T cells binding during electrophoretic mobility shift assay analysis and circumvented the HIF-1alpha mRNA increase. Knockdown experiments, receptor analysis, and antibody neutralization pointed to sphingosine-1-phosphate and transforming growth factor-beta as the initiators of the HIF-1 response. The use of macrophages from conditional HIF-1alpha knockout mice revealed that macrophages, under the impact of apoptotic cell supernatants, use HIF-1 to produce factors that induce CD31 expression in murine embryonic stem cells. Our study supports the notion that soluble factors produced from apoptotic tumor cells activate the HIF-1 system under normoxia in macrophages to enhance their tumor-promoting capacity by, for example, releasing vascular endothelial growth factor. This shows the importance of HIF-1-elicited responses in regulatory macrophages under normoxia.

A23187, ionomycin and thapsigargin upregulate mRNA of HIF-1alpha via endoplasmic reticulum stress rather than a rise in intracellular calcium.

Hypoxia inducible factor 1 (HIF-1) coordinates major responses to hypoxia, with the notion that its alpha subunit is degraded under normoxia but stable under hypoxia. Recently, calcium was shown to affect expression of HIF-1alpha. While lowering cytosolic calcium accumulates HIF-1alpha, a calcium increase by the ionophores A23187 or ionomycin as well as the endoplasmic reticulum (ER) Ca(2+)-ATPase inhibitor thapsigargin produced inconsistent results with reports to either increase or decrease HIF-1alpha protein. In the human hepatocyte cell line HepG2 only A23187, but neither ionomycin nor thapsigargin, accumulated HIF-1alpha protein under normoxia. However, A23187 does so independently of a calcium increase. A23187 not only accumulated HIF-1alpha protein but also mRNA of HIF-1alpha, with the notion that protein but not mRNA accumulation was attenuated by blocking mitogen-activated protein kinase (MAPK), suggesting that HIF-1alpha protein accumulation is not a direct consequence of its mRNA elevation. Indeed, protein stability determinations implied that A23187 enhanced translation of HIF-1alpha. Interestingly, ionomycin and thapsigargin also increased the HIF-1alpha mRNA content. Although not increasing the HIF-1alpha protein amount under normoxia, both compounds enhanced protein accumulation of HIF-1alpha under hypoxia. Taking into account that induction of ER stress by tunicamycin and brefeldin A, without altering intracellular calcium concentrations, also increased HIF-1alpha mRNA, suggests that ER stress pathways enhanced transcription of HIF-1alpha mRNA. We conclude that ER stress rather than calcium fluctuations increased HIF-1alpha mRNA content by established calcium liberating agents, which alone is insufficient for normoxic HIF-1alpha accumulation.