Primary Tumor Resection Decelerates Disease Progression in an Orthotopic Mouse Model of Metastatic Prostate Cancer

Radical prostatectomy in oligometastatic prostate cancer is a matter of intense debate. Besides avoiding local complications, it is hypothesized that primary tumor resection may result in better oncological outcomes. The aim of our study was to analyze the effect of primary tumor resection on disease progression in an orthotopic prostate cancer mouse model. First, the optimal time point for primary tumor resection, when metastases have already occurred, but the primary tumor is still resectable, was determined as 8 weeks after inoculation of 5 × 105 LuCaP136 cells. In a second in vivo experiment, 64 mice with metastatic prostate cancer were randomized into two groups, primary tumor resection or sham operation, and disease progression was followed up for 10 weeks. The technique of orthotopic primary tumor resection was successfully established. Compared with the sham operation group, mice with primary tumor resection showed a significantly longer survival (p < 0.001), a significantly slower PSA increase (p < 0.01), and a lower number of lung metastases (p = 0.073). In conclusion, primary tumor resection resulted in slower disease progression and longer survival in an orthotopic mouse model of metastatic prostate cancer. In future studies, this model will be used to unravel the molecular mechanisms of primary tumor/metastasis interaction in prostate cancer

Cancer-associated fibroblasts stimulate primary tumor growth and metastatic spread in an orthotopic prostate cancer xenograft model

The unique microenvironment of the prostate plays a crucial role in the development and progression of prostate cancer (PCa). We examined the effects of cancer-associated fibroblasts (CAFs) on PCa progression using patient-derived fibroblast primary cultures in a representative orthotopic xenograft model. Primary cultures of CAFs, non-cancer-associated fibroblasts (NCAFs) and benign prostate hyperplasia-associated fibroblasts (BPHFs) were generated from patient-derived tissue specimens. These fibroblasts were coinjected together with cancer cells (LuCaP136 spheroids or LNCaP cells) in orthotopic PCa xenografts to investigate their effects on local and systemic tumor progression. Primary tumor growth as well as metastatic spread to lymph nodes and lungs were significantly stimulated by CAF coinjection in LuCaP136 xenografts. When NCAFs or BPHFs were coinjected, tumor progression was similar to injection of tumor cells alone. In LNCaP xenografts, all three fibroblast types significantly stimulated primary tumor progression compared to injection of LNCaP cells alone. CAF coinjection further increased the frequency of lymph node and lung metastases. This is the first study using an orthotopic spheroid culture xenograft model to demonstrate a stimulatory effect of patient-derived CAFs on PCa progression. The established experimental setup will provide a valuable tool to further unravel the interacting mechanisms between PCa cells and their microenvironment

Regional specialization and fate specification of bone stromal cells in skeletal development

Bone stroma contributes to the regulation of osteogenesis and hematopoiesis but also to fracture healing and disease processes. Mesenchymal stromal cells from bone (BMSCs) represent a heterogenous mixture of different subpopulations with distinct molecular and functional properties. The lineage relationship between BMSC subsets and their regulation by intrinsic and extrinsic factors are not well understood. Here, we show with mouse genetics, ex vivo cell differentiation assays, and transcriptional profiling that BMSCs from metaphysis (mpMSCs) and diaphysis (dpMSCs) are fundamentally distinct. Fate-tracking experiments and single-cell RNA sequencing indicate that bone-forming osteoblast lineage cells and dpMSCs, including leptin receptor-positive (LepR(+)) reticular cells in bone marrow, emerge from mpMSCs in the postnatal metaphysis. Finally, we show that BMSC fate is controlled by platelet-derived growth factor receptor β (PDGFRβ) signaling and the transcription factor Jun-B. The sum of our findings improves our understanding of BMSC development, lineage relationships, and differentiation

Multibudded tubules formed by COPII on artificial liposomes

COPII-coated vesicles form at the endoplasmic reticulum for cargo transport to the Golgi apparatus. We used in vitro reconstitution to examine the roles of the COPII scaffold in remodeling the shape of a lipid bilayer. Giant Unilamellar Vesicles were examined using fast confocal fluorescence and cryo-electron microscopy in order to avoid separation steps and minimize mechanical manipulation. COPII showed a preference for high curvature structures, but also sufficient flexibility for binding to low curvatures. The COPII proteins induced beads-on-a-string-like constricted tubules, similar to those previously observed in cells. We speculate about a mechanical pathway for vesicle fission from these multibudded COPII-coated tubules, considering the possibility that withdrawal of the Sar1 amphipathic helix upon GTP hydrolysis leads to lipid bilayer destabilization resulting in fission

Is the 6 kDa tobacco etch viral protein a bona fide ERES marker?

The claim that the 6 kDa viral protein (VP) of Tobacco Etch Virus is a marker for ER exit sites (ERES) has been investigated. When transiently expressed as a CFP tagged fusion construct in tobacco mesophyll protoplasts, this integral membrane protein co-localizes with both the COPII coat protein YFP-SEC24 and the Golgi marker Man1-RFP. However, when over-expressed the VP locates to larger spherical structures which co-localize with neither ER nor Golgi markers. Nevertheless, deletion of the COPII interactive N-terminal D(X)E motif causes it to be broadly distributed throughout the ER, supporting the notion that this protein could be an ERES marker. Curiously, whereas brefeldin A (BFA) caused a typical Golgi-stack response (redistribution into the ER) of the VP in leaf epidermal cells, in protoplasts it resulted in the formation of structures identical to those formed by over-expression. However, anomalous results were obtained with protoplasts: when co-expressed with the non-cycling cis-Golgi marker Man1-RFP, a BFA-induced redistribution of the VP-CFP signal into the ER was observed, but, in the presence of the cycling Golgi marker ERD2-YFP, this did not occur. High resolution images of side-on views of Golgi stacks in epidermal cells showed that the 6 kDa VP-CFP signal overlapped considerably more with YFP-SEC24 than with Man1-RFP, indicating that the VP is proportionately more associated with ERES. However, based on a consideration of the structure of its cytoplasmic tail, the scenario that the VP collects at ERES and is transported to the cis-Golgi before being recycled back to the ER, is supported

Toward visualization of nanomachines in their native cellular environment

The cellular nanocosm is made up of numerous types of macromolecular complexes or biological nanomachines. These form functional modules that are organized into complex subcellular networks. Information on the ultra-structure of these nanomachines has mainly been obtained by analyzing isolated structures, using imaging techniques such as X-ray crystallography, NMR, or single particle electron microscopy (EM). Yet there is a strong need to image biological complexes in a native state and within a cellular environment, in order to gain a better understanding of their functions. Emerging methods in EM are now making this goal reachable. Cryo-electron tomography bypasses the need for conventional fixatives, dehydration and stains, so that a close-to-native environment is retained. As this technique is approaching macromolecular resolution, it is possible to create maps of individual macromolecular complexes. X-ray and NMR data can be ‘docked’ or fitted into the lower resolution particle density maps to create a macromolecular atlas of the cell under normal and pathological conditions. The majority of cells, however, are too thick to be imaged in an intact state and therefore methods such as ‘high pressure freezing’ with ‘freeze-substitution followed by room temperature plastic sectioning’ or ‘cryo-sectioning of unperturbed vitreous fully hydrated samples’ have been introduced for electron tomography. Here, we review methodological considerations for visualizing nanomachines in a close-to-physiological, cellular context. EM is in a renaissance, and further innovations and training in this field should be fully supported

Gingival Fibroblasts as a Promising Source of Induced Pluripotent Stem Cells

Induced pluripotent stem (iPS) cells efficiently generated from accessible tissues have the potential for clinical applications. Oral gingiva, which is often resected during general dental treatments and treated as biomedical waste, is an easily obtainable tissue, and cells can be isolated from patients with minimal discomfort.We herein demonstrate iPS cell generation from adult wild-type mouse gingival fibroblasts (GFs) via introduction of four factors (Oct3/4, Sox2, Klf4 and c-Myc; GF-iPS-4F cells) or three factors (the same as GF-iPS-4F cells, but without the c-Myc oncogene; GF-iPS-3F cells) without drug selection. iPS cells were also generated from primary human gingival fibroblasts via four-factor transduction. These cells exhibited the morphology and growth properties of embryonic stem (ES) cells and expressed ES cell marker genes, with a decreased CpG methylation ratio in promoter regions of Nanog and Oct3/4. Additionally, teratoma formation assays showed ES cell-like derivation of cells and tissues representative of all three germ layers. In comparison to mouse GF-iPS-4F cells, GF-iPS-3F cells showed consistently more ES cell-like characteristics in terms of DNA methylation status and gene expression, although the reprogramming process was substantially delayed and the overall efficiency was also reduced. When transplanted into blastocysts, GF-iPS-3F cells gave rise to chimeras and contributed to the development of the germline. Notably, the four-factor reprogramming efficiency of mouse GFs was more than 7-fold higher than that of fibroblasts from tail-tips, possibly because of their high proliferative capacity.These results suggest that GFs from the easily obtainable gingival tissues can be readily reprogrammed into iPS cells, thus making them a promising cell source for investigating the basis of cellular reprogramming and pluripotency for future clinical applications. In addition, high-quality iPS cells were generated from mouse GFs without Myc transduction or a specific system for reprogrammed cell selection

Mitochondrial Physiology and Gene Expression Analyses Reveal Metabolic and Translational Dysregulation in Oocyte-Induced Somatic Nuclear Reprogramming

While reprogramming a foreign nucleus after somatic cell nuclear transfer (SCNT), the enucleated oocyte (ooplasm) must signal that biomass and cellular requirements changed compared to the nucleus donor cell. Using cells expressing nuclear-encoded but mitochondria-targeted EGFP, a strategy was developed to directly distinguish maternal and embryonic products, testing ooplasm demands on transcriptional and post-transcriptional activity during reprogramming. Specifically, we compared transcript and protein levels for EGFP and other products in pre-implantation SCNT embryos, side-by-side to fertilized controls (embryos produced from the same oocyte pool, by intracytoplasmic injection of sperm containing the EGFP transgene). We observed that while EGFP transcript abundance is not different, protein levels are significantly lower in SCNT compared to fertilized blastocysts. This was not observed for Gapdh and Actb, whose protein reflected mRNA. This transcript-protein relationship indicates that the somatic nucleus can keep up with ooplasm transcript demands, whilst transcription and translation mismatch occurs after SCNT for certain mRNAs. We further detected metabolic disturbances after SCNT, suggesting a place among forces regulating post-transcriptional changes during reprogramming. Our observations ascribe oocyte-induced reprogramming with previously unsuspected regulatory dimensions, in that presence of functional proteins may no longer be inferred from mRNA, but rather depend on post-transcriptional regulation possibly modulated through metabolism