Presenting embryologically defined peripancreatic compartments and fusion planes in the search for pancreatic cancer fields.

BACKGROUND: Clinical progress in form of "total mesometrial resection" (TMMR) in cervical cancer and "total mesorectal excision" (TME) in rectal cancer can be traced to a paradigm-shift regarding the extent and range of resection. More significance is bestowed upon embryologically defined borders which define compartments, "morphogenetic units" and "cancer fields", that have to be addressed in order to avoid incomplete tumor resection. We want to transfer this rationale on the pancreas and define such borders for pancreatic compartments. MATERIAL AND METHODS: We used 26 unfixed body donors (16 male, 10 female) ranging in age from 64 to 98 years. Manual preparation consisted of performing the Cattell-Braasch maneuver to restore embryologic anatomy and define fascial remnants of the borders of the dorsal and ventral mesogastrium with focus on the pancreatic fusion fasciae and peripancreatic spaces. RESULTS: We tracked what used to be the dorsal and ventral mesogastrium and assigned their remnants to the bowel and pancreas. Following avascular embryologic fascial fusion planes along the mesogastria we could demonstrate peripancreatic spaces, which were sealed off from bordering surfaces of presumably different morphogenetic units and possible cancer fields. Reverting embryologic development also seemed possible within the pancreas, demonstrating the embryologic fusion plane between the ventral and dorsal pancreatic buds as two distinct compartments. CONCLUSIONS: Following pancreatic fusion fasciae by separating embryologic fusion planes enables to define the pancreatic compartments which might play a major role in applying the success of TMMR and TME on pancreatic resection and define pancreatic cancer fields.

RNA sequencing of glioblastoma tissue slice cultures reveals the effects of treatment at the transcriptional level.

One of the major challenges in cancer research is finding models that closely resemble tumors within patients. Human tissue slice cultures are a promising approach to provide a model of the patient's tumor biology ex vivo. Recently, it was shown that these slices can be successfully analyzed by whole transcriptome sequencing as well as automated histochemistry, increasing their usability as preclinical model. Glioblastoma multiforme (GBM) is a highly malignant brain tumor with poor prognosis and little is known about its genetic background and heterogeneity regarding therapy success. In this study, tissue from the tumors of 25 patients with primary GBM was processed into slice cultures and treated with standard therapy (irradiation and temozolomide). Total RNA sequencing and automated histochemistry were performed to enable analysis of treatment effects at a transcriptional and histological level. Slice cultures from long-term survivors (overall survival [OS] > 24 months) exhibited more apoptosis than cultures from patients with shorter OS. Proliferation within these slices was slightly increased in contrast to other groups, but not significantly. Among all samples, 58 protein-coding genes were upregulated and 32 downregulated in treated vs. untreated slice cultures. In general, an upregulation of DNA damage-related and cell cycle checkpoint genes as well as enrichment of genotoxicity pathways and p53-dependent signaling was found after treatment. Overall, the current study reproduces knowledge from former studies regarding the feasibility of transcriptomic analyses and automated histology in tissue slice cultures. We further demonstrate that the experimental data merge with the clinical follow-up of the patients, which improves the applicability of our model system.

Leptin Receptor-Deficient db/db Mice Show Significant Heterogeneity in Response to High Non-heme Iron Diet.

Recent studies have shown an association between iron homeostasis, obesity and diabetes. In this work, we investigated the differences in the metabolic status and inflammation in liver, pancreas and visceral adipose tissue of leptin receptor-deficient db/db mice dependent on high iron concentration diet. 3-month-old male BKS-Leprdb/db/JOrlRj (db/db) mice were divided into two groups, which were fed with different diets containing high iron (29 g/kg, n = 57) or standard iron (0.178 g/kg; n = 42) concentrations for 4 months. As anticipated, standard iron-fed db/db mice developed obesity and diabetes. However, high iron-fed mice exhibited a wide heterogeneity. By dividing into two subgroups at the diabetes level, non-diabetic subgroup 1 (<13.5 mmol/l, n = 30) significantly differed from diabetic subgroup two (>13.5 mmol/l, n = 27). Blood glucose concentration, HbA1c value, inflammation markers interleukin six and tumor necrosis factor α and heme oxygenase one in visceral adipose tissue were reduced in subgroup one compared to subgroup two. In contrast, body weight, C-peptide, serum insulin and serum iron concentrations, pancreatic islet and signal ratio as well as cholesterol, LDL and HDL levels were enhanced in subgroup one. While these significant differences require further studies and explanation, our results might also explain the often-contradictory results of the metabolic studies with db/db mice.

Human tissue cultures of lung cancer predict patient susceptibility to immune-checkpoint inhibition.

Despite novel immunotherapies being approved and established for the treatment of non-small cell lung cancer (NSCLC), ex vivo models predicting individual patients' responses to immunotherapies are missing. Especially immune modulating therapies with moderate response rates urge for biomarkers and/or assays to determine individual prediction of treatment response and investigate resistance mechanisms. Here, we describe a standardized ex vivo tissue culture model to investigate individual tumor responses. NSCLC tissue cultures preserve morphological characteristics of the baseline tumor specimen for up to 12 days ex vivo and also maintain T-cell function for up to 10 days ex vivo. A semi-automated analysis of proliferating and apoptotic tumor cells was used to evaluate tissue responses to the PD-1 inhibitor nivolumab (n = 12), from which two cases could be successfully correlated to the clinical outcome. T-cell responses upon nivolumab treatment were investigated by flow cytometry and multispectral imaging. Alterations in the frequency of the Treg population and reorganization of tumor tissues could be correlated to nivolumab responsiveness ex vivo. Thus, our findings not only demonstrate the functionality of T cells in NSCLC slice cultures up to 10 days ex vivo, but also suggests this model for stratifying patients for treatment selection and to investigate in depth the tumor-associated T-cell regulation.

PD-1 inhibition in patient derived tissue cultures of human gastric and gastroesophageal adenocarcinoma.

Emerging immunotherapies quest for better patient stratification in cancer treatment decisions. Moderate response rates of PD-1 inhibition in gastric and esophagogastric junction cancers urge for meaningful human model systems that allow for investigating immune responses ex vivo. Here, the standardized patient-derived tissue culture (PDTC) model was applied to investigate tumor response to the PD-1 inhibitor Nivolumab and the CD3/CD28 t-lymphocyte activator ImmunoCultTM. Resident t-lymphocytes, tumor proliferation and apoptosis, as well as bulk gene expression data were analyzed after 72 h of PD-1 inhibition either as monotherapy or combined with Oxaliplatin or ImmunoCultTM. Individual responses to PD-1 inhibition were found ex vivo and combination with chemotherapy or t-lymphocyte activation led to enhanced antitumoral effects in PDTCs. T-lymphocyte activation as well as the addition of pre-cultured peripheral blood mononuclear cells improved PDTC for studying t-lymphocyte and tumor cell communication. These data support the potential of PDTC to investigate immunotherapy ex vivo in gastric and esophagogastric junction cancer.

Adipocyte death triggers a pro-inflammatory response and induces metabolic activation of resident macrophages.

A chronic low-grade inflammation within adipose tissue (AT) seems to be the link between obesity and some of its associated diseases. One hallmark of this AT inflammation is the accumulation of AT macrophages (ATMs) around dead or dying adipocytes, forming so-called crown-like structures (CLS). To investigate the dynamics of CLS and their direct impact on the activation state of ATMs, we established a laser injury model to deplete individual adipocytes in living AT from double reporter mice (GFP-labeled ATMs and tdTomato-labeled adipocytes). Hence, we were able to detect early ATM-adipocyte interactions by live imaging and to determine a precise timeline for CLS formation after adipocyte death. Further, our data indicate metabolic activation and increased lipid metabolism in ATMs upon forming CLS. Most importantly, adipocyte death, even in lean animals under homeostatic conditions, leads to a locally confined inflammation, which is in sharp contrast to other tissues. We identified cell size as cause for the described pro-inflammatory response, as the size of adipocytes is above a critical threshold size for efferocytosis, a process for anti-inflammatory removal of dead cells during tissue homeostasis. Finally, experiments on parabiotic mice verified that adipocyte death leads to a pro-inflammatory response of resident ATMs in vivo, without significant recruitment of blood monocytes. Our data indicate that adipocyte death triggers a unique degradation process and locally induces a metabolically activated ATM phenotype that is globally observed with obesity.

FOXM1 Inhibition in Ovarian Cancer Tissue Cultures Affects Individual Treatment Susceptibility Ex Vivo.

Diagnosis in an advanced state is a major hallmark of ovarian cancer and recurrence after first line treatment is common. With upcoming novel therapies, tumor markers that support patient stratification are urgently needed to prevent ineffective therapy. Therefore, the transcription factor FOXM1 is a promising target in ovarian cancer as it is frequently overexpressed and associated with poor prognosis. In this study, fresh tissue specimens of 10 ovarian cancers were collected to investigate tissue cultures in their ability to predict individual treatment susceptibility and to identify the benefit of FOXM1 inhibition. FOXM1 inhibition was induced by thiostrepton (3 µM). Carboplatin (0.2, 2 and 20 µM) and olaparib (10 µM) were applied and tumor susceptibility was analyzed by tumor cell proliferation and apoptosis in immunofluorescence microscopy. Resistance mechanisms were investigated by determining the gene expression of FOXM1 and its targets BRCA1/2 and RAD51. Ovarian cancer tissue was successfully maintained for up to 14 days ex vivo, preserving morphological characteristics of the native specimen. Thiostrepton downregulated FOXM1 expression in tissue culture. Individual responses were observed after combined treatment with carboplatin or olaparib. Thus, we successfully implemented a complex tissue culture model to ovarian cancer and showed potential benefit of combined FOXM1 inhibition.

CD4+ T cells regulate glucose homeostasis independent of adipose tissue dysfunction in mice.

Obesity is frequently associated with a chronic low-grade inflammation in the adipose tissue (AT) and impaired glucose homeostasis. Adipose tissue macrophages (ATMs) have been shown to accumulate in the inflamed AT either by means of recruitment from the blood or local proliferation. ATM proliferation and activation can be stimulated by TH2 cytokines, such as IL-4 and IL-13, suggesting involvement of CD4-positive T cells in ATM proliferation and activation. Furthermore, several studies have associated T cells to alterations in glucose metabolism. Therefore, we sought to examine a direct impact of CD4-positive T cells on ATM activation, ATM proliferation and glucose homeostasis using an in vivo depletion model. Surprisingly, CD4 depletion did not affect ATM activation, ATM proliferation, or insulin sensitivity. However, CD4 depletion led to a significant improvement of glucose tolerance. In line with this, we found moderate disturbances in pancreatic endocrine function following CD4 depletion. Hence, our data suggest that the effect on glucose metabolism observed after CD4 depletion might be mediated by organs other than AT and independent of AT inflammation.

[Is there a new salivary gland? - Rather not!] / Gibt es eine neue Kopfspeicheldrüse? ­ Eher nicht!

In October 2020, the lay press, but also some medical journals and websites reported the putative discovery of a new salivary gland in the nasopharynx based on prostate-specific membrane antigen positron emission tomography computed tomography (PSMA-PET/CT) examinations. As an interdisciplinary group from the fields of anatomy, pathology, nuclear medicine and otorhinolaryngology, we come to the view that an accumulation of minor salivary glands has been described here. Minor salivary glands in the nasopharynx and in the peritubar region have been described at least since 1866. The current description in PSMA-PET/CT does not justify the definition of a new, independent salivary gland. The PSMA-PET/CT could, however, be suitable to better protect salivary glands in the nasopharynx when planning radiation therapy. This should be evaluated in clinical trials.

Fast microglial activation after severe traumatic brain injuries.

Traumatic brain injury is among the leading causes of death in individuals under 45 years of age. However, since trauma mechanisms and survival times differ enormously, the exact mechanisms leading to the primary and secondary injury and eventually to death after traumatic brain injury (TBI) remain unclear. Several studies showed the versatile functions of microglia, the innate macrophages of the brain, following a TBI. Earlier being characterized as rather neurotoxic, neuroprotective capacities were recently demonstrated, therefore, making microglia one of the key players following TBI. Especially in cases with only short survival times, immediate microglial reactions are of great forensic interest in questions of wound age estimation. Using standardized immunohistochemical methods, we examined 8 cases which died causatively of TBI with survival times between minutes and 7 days and 5 control cases with cardiovascular failure as the cause of death to determine acute changes in microglial morphology and antigen expression after TBI. In this pilot study, we detected highly localized changes in microglial morphology already early after traumatic damage, e.g., activated microglia and phagocyted erythrocytes in the contusion areas in cases with minute survival. Furthermore, an altered antigen expression was observed with increasing trauma wound age, showing similar effects like earlier transcriptomic studies. There is minute data on the direct impact of shear forces on microglial morphology. We were able to show localization-depending effects on microglial morphology causing localized dystrophy and adjacent activation. While rodent studies are widespread, they fail to mimic the exact mechanisms in human TBI response. Therefore, more studies focusing on cadaveric samples need to follow to thoroughly define the mechanisms leading to cell destruction and eventually evaluate their forensic value.

Examination of ex-vivo viability of human adipose tissue slice culture.

Obesity is associated with significantly higher mortality rates, and excess adipose tissue is involved in respective pathologies. Here we established a human adipose tissue slice cultures (HATSC) model ex vivo. HATSC match the in vivo cell composition of human adipose tissue with, among others, mature adipocytes, mesenchymal stem cells as well as stroma tissue and immune cells. This is a new method, optimized for live imaging, to study adipose tissue and cell-based mechanisms of obesity in particular. HATSC survival was tested by means of conventional and immunofluorescence histological techniques, functional analyses and live imaging. Surgery-derived tissue was cut with a tissue chopper in 500 µm sections and transferred onto membranes building an air-liquid interface. HATSC were cultured in six-well plates filled with Dulbecco's Modified Eagle's Medium (DMEM), insulin, transferrin, and selenium, both with and without serum. After 0, 1, 7 and 14 days in vitro, slices were fixated and analyzed by morphology and Perilipin A for tissue viability. Immunofluorescent staining against IBA1, CD68 and Ki67 was performed to determine macrophage survival and proliferation. These experiments showed preservation of adipose tissue as well as survival and proliferation of monocytes and stroma tissue for at least 14 days in vitro even in the absence of serum. The physiological capabilities of adipocytes were functionally tested by insulin stimulation and measurement of Phospho-Akt on day 7 and 14 in vitro. Viability was further confirmed by live imaging using Calcein-AM (viable cells) and propidium iodide (apoptosis/necrosis). In conclusion, HATSC have been successfully established by preserving the monovacuolar form of adipocytes and surrounding macrophages and connective tissue. This model allows further analysis of mature human adipose tissue biology ex vivo.

Unspecific DNA recombination in AdipoqCre-ERT2 - mediated knockout approaches in transgenic mice is sex-, age- and genotype-dependent.

Due to the epidemic rise of obesity prevalence, adipose tissue (AT) research is of major interest. Our aim was to study specificity of the most-common Cre/loxP approach for inducible gene manipulation of AT in mice (AdipoqCre-ERT2). We used mice with tamoxifen-sensitive Cre recombinase controlled by the adiponectin promoter (AdipoqCre-ERT2), which were crossed to a tdTomato reporter mouse to visualize the site of recombination on a single-cell resolution. Albeit tamoxifen induced tdTomato expression in this model, also non-stimulated background recombination ('Cre leakage') was detected in AT of untreated Adipoq-CreERT2xTDTO mice in vivo. Quantification of Cre leakage revealed age, sex and genotype as factors impacting on non-induced Cre recombination.

Deep sequencing and automated histochemistry of human tissue slice cultures improve their usability as preclinical model for cancer research.

Cancer research requires models closely resembling the tumor in the patient. Human tissue cultures can overcome interspecies limitations of animal models or the loss of tissue architecture in in vitro models. However, analysis of tissue slices is often limited to histology. Here, we demonstrate that slices are also suitable for whole transcriptome sequencing and present a method for automated histochemistry of whole slices. Tumor and peritumoral tissue from a patient with glioblastoma was processed to slice cultures, which were treated with standard therapy including temozolomide and X-irradiation. Then, RNA sequencing and automated histochemistry were performed. RNA sequencing was successfully accomplished with a sequencing depth of 243 to 368 x 106 reads per sample. Comparing tumor and peritumoral tissue, we identified 1888 genes significantly downregulated and 2382 genes upregulated in tumor. Treatment significantly downregulated 2017 genes, whereas 1399 genes were upregulated. Pathway analysis revealed changes in the expression profile of treated glioblastoma tissue pointing towards downregulated proliferation. This was confirmed by automated analysis of whole tissue slices stained for Ki67. In conclusion, we demonstrate that RNA sequencing of tissue slices is possible and that histochemical analysis of whole tissue slices can be automated which increases the usability of this preclinical model.

Tumor-associated macrophages and individual chemo-susceptibility are influenced by iron chelation in human slice cultures of gastric cancer.

Purpose: Presence of tumor-associated macrophages (TAM) and high levels of ferritin and lipocalin 2 (Lcn2) in the tumor microenvironment are associated with poor prognosis in many types of cancer. Here we investigate whether iron deprivation influences TAM phenotype and chemotherapy resistance in tumor slice cultures (TSC) of gastric cancer. Results: TAM remained morphologically and functionally stable for four DIV. DFO treatment for 72 h decreased ferritin expression in TAM and in the tumor stroma but did not alter Lcn2 expression. TAM phenotype was altered after 72 h of cisplatin or DFO treatment compared with control conditions. Single DFO treatment and combined treatment with cytotoxic drugs significantly increased tumor cell apoptosis in TSC of gastric cancer. Methods: TSC were manufactured by cutting tissue of gastric cancer resection specimens in 350 µm thick slices and cultivating them under standard conditions on a filter membrane, at an air-liquid interface. After 24 h ex vivo, TSC were treated with irinotecan (100 nM) or cisplatin (10 µM) alone and in combination with deferoxamine (DFO; 10 µM, 100 µM), respectively, for 72 h. After four days in vitro (DIV) the TSC were fixated with paraformaldehyde, paraffin embedded and analyzed by immunohistochemistry for apoptosis (cPARP), proliferation (Ki67), TAM (CD68, CD163), ferritin, and Lcn2 expression. Conclusions: TAM are well preserved and can be studied in TSC of gastric cancer. Iron deprivation significantly increased tumor cell apoptosis.

NRG1 type I dependent autoparacrine stimulation of Schwann cells in onion bulbs of peripheral neuropathies.

In contrast to acute peripheral nerve injury, the molecular response of Schwann cells in chronic neuropathies remains poorly understood. Onion bulb structures are a pathological hallmark of demyelinating neuropathies, but the nature of these formations is unknown. Here, we show that Schwann cells induce the expression of Neuregulin-1 type I (NRG1-I), a paracrine growth factor, in various chronic demyelinating diseases. Genetic disruption of Schwann cell-derived NRG1 signalling in a mouse model of Charcot-Marie-Tooth Disease 1A (CMT1A), suppresses hypermyelination and the formation of onion bulbs. Transgenic overexpression of NRG1-I in Schwann cells on a wildtype background is sufficient to mediate an interaction between Schwann cells via an ErbB2 receptor-MEK/ERK signaling axis, which causes onion bulb formations and results in a peripheral neuropathy reminiscent of CMT1A. We suggest that diseased Schwann cells mount a regeneration program that is beneficial in acute nerve injury, but that overstimulation of Schwann cells in chronic neuropathies is detrimental.

The Role of Iron and Nerve Inflammation in Diabetes Mellitus Type 2-Induced Peripheral Neuropathy.

Peripheral diabetic neuropathy (PDN) is one of the most common complications of diabetes mellitus. Previous studies showed an association between dietary iron load and inflammation in the development of PDN in a rat model of type 1 diabetes (T1D). Here we investigated the role of iron and neural inflammation in development of PDN in a animal model of obesity and type 2 diabetes (T2D). 3-month-old db/db mice were fed with a high, standard or low iron diet for 4 months. High iron chow lead to a significant increase in motor nerve conduction velocities compared to mice on standard and low iron chow. Direct beneficiary effects on lowering blood glucose and HbA1c concentrations were shown in the high iron treated diabetic mice. Numbers of pro-inflammatory M1 macrophages were reduced in nerve sections, and anti-inflammatory M2 macrophages were increased in db/db mice on high iron diet compared to other groups. These results confirm and extend our previous findings in STZ-diabetic rats by showing that dietary non-hem iron supplementation may partly prevent the development of PDN in opposition to iron restriction. The identification of these dietary iron effects on the metabolic and inflammatory mechanisms of PDN supports a role of dietary iron and leads us to suggest testing for iron levels in human diabetic patients.

The role of dietary non-heme iron load and peripheral nerve inflammation in the development of peripheral neuropathy (PN) in obese non-diabetic leptin-deficient ob/ob mice.

INTRODUCTION: Here, we investigated inflammatory signs of peripheral nerves in leptin-deficient obese ob/ob mice and the modulating effects of the exogenous iron load. METHODS: Ob/ob and ob/+ control mice were fed with high, standard, or low iron diet for four months. RESULTS: We found intraepidermal nerve fiber degeneration in foot skin and low-grade neuropathic abnormalities including mildly slowed motor and compound sensory nerve conduction velocities and low-grade macrophage and T-cell infiltration without overt neuropathology in sciatic nerves of all ob/ob mice. Low dietary iron load caused more pronounced abnormalities than high iron load in ob/ob mice. DISCUSSION: Our data suggest that dietary non-heme iron deficiency may be a modulating factor in the pathogenesis of peripheral neuropathy in obese ob/ob mice with metabolic syndrome. Once the mechanisms can be further elucidated, how low dietary iron augments peripheral nerve degeneration and dysfunction via pro-inflammatory pathways and new therapeutic strategies could be developed. ABBREVIATIONS: CMAP: compound muscle action potential; cSNCV: compound sensory nerve conduction velocity; IENFD: intraepidermal nerve fiber density; LDL: low-density lipoprotein; MetS: metabolic syndrome; MNCV: motor conduction velocity; NCV: nerve conduction velocity; PN: peripheral neuropathy; PNS: peripheral nervous system; STZ: streptozotocin; T2D: type 2 diabetes mellitus; TNF alpha: tumor necrosis factor alpha; WHO: World Health Organization.

Impact of X-irradiation on microglia.

Irradiation is widely used to treat brain tumors, and also to create bone marrow (BM) chimeras. BM chimeras are widely used to dissect functions and origin of microglia and blood-derived mononuclear cells under homeostatic or pathological conditions. This is facilitated by the fact that microglia survive irradiation and are thus regarded radio-resistant. In this study, we tested whether microglia are indeed radio-resistant and looked for potential mechanisms that might explain this phenomenon. We analyzed the radio-resistance of microglia independently of their physiological brain environment compared to other mononuclear cells from spleen and brain after X-irradiation with 7 Gy or 30 Gy. Furthermore, we investigated long-term effects of X-irradiation on microglia using organotypic hippocampal slice cultures (OHSCs). We found a significant higher survival rate of isolated microglia 4 hr after X-irradiation with 30 Gy accompanied by a decreased proliferation rate. Investigations of apoptosis-related genes revealed no regulation of a specific antiapoptotic pathway but ataxia telangiectasia mutated (ATM), a DNA-repair-related gene, was significantly upregulated in isolated microglia 4 hr after 30 Gy. Irradiation of OHSCs with 7 and 30 Gy revealed a highly and significantly decreased cell number, morphological changes and an increase in migration velocity of microglia. Furthermore, cell loss, increased soma size and process length of microglia was also found in BM chimeras irradiated with 9.5 Gy 5 weeks after irradiation. Here, we present new evidence implying that microglia are not a homogeneous population of radio-resistant cells and report on long-term alterations of microglia that survived irradiation.

Individual Susceptibility Analysis Using Patient-derived Slice Cultures of Colorectal Carcinoma.

BACKGROUND: Nonresponse to chemotherapy in colorectal carcinoma (CRC) is still a clinical problem. For most established treatment regimens, no predictive biomarkers are available. Patient-derived tumor slice culture may be a promising ex vivo technology to assess the drug susceptibility in individual tumors. METHODS: Patient-derived slice cultures of CRC specimens were prepared according to a standardized protocol and treated with different concentrations of 5-fluorouracil (5-FU) and an adapted FOLFOX regimen (5-FU and oxaliplatin) to investigate histologic response. Additionally, a semi-automatized readout using fluorescent stain-specific segmentation algorithms for Image J was established to quantify changes in tumor proliferation. Nonresponse to chemotherapy was defined as persisting tumor cell proliferation. RESULTS: Slices treated with 5-FU showed lower tumor cell fractions and dose-dependent alterations of proliferating tumor cells compared with controls (1 µM, Δ +3%; 10 µM, Δ -9%; 100 µM, Δ -15%). Individual tumor samples were examined and differences in chemotherapy susceptibility could be observed. Untreated slice cultures contained an average tumor cell fraction of 31% ± 7%. For all samples, the histopathologic characteristics exhibited some degree of intratumoral heterogeneity with regard to tumor cell morphology and distribution. The original tumor matched the features found in slices at baseline and after 3 days of cultivation. CONCLUSIONS: Patient-derived slice cultures may help to predict response to clinical treatment in individual patients with CRC. Future studies need to address the problem of tumor heterogeneity and evolution. Prospective correlation of ex vivo results with the clinical course of treated patients is warranted.

Neuroanatomy of pain-deficiency and cross-modal activation in calcium channel subunit (CACN) α2δ3 knockout mice.

The phenotype of calcium channel subunit (CACN) α2δ3 knockout (KO) mice includes sensory cross-activation and deficient pain perception. Sensory cross-activation defines the activation of a sensory cortical region by input from another modality due to reorganization in the brain such as after sensory loss. To obtain mechanistic insight into both phenomena, we employed a comprehensive battery of neuroanatomical techniques. While CACNα2δ3 was ubiquitously expressed in wild-type mice, it was absent in α2δ3 KO animals. Immunostaining of α1A, α1B, and α1E revealed upregulation of N-type and R-type, but not P/Q-type Cav2 channels in cortical neurons of CACNα2δ3 KO mice. Compared to wild-type mice, axonal processes in somatosensory cortex were enhanced, and dendritic processes reduced, in CACNα2δ3 KO mice. Immunohistochemical and MRI analyses, investigating morphology, thalamocortical and intra-/intercortical trajectories, revealed a disparity between projection and commissural fibers with reduction of the number of spatial specificity of thalamocortical projections. L1cam staining revealed wide-ranging projections of thalamocortical fibers reaching both somatosensory/motor and visual cortical areas. Activation (c-fos+) of excitatory and inhibitory neurons suggested that deficient pain perception in α2δ3 KO mice is unlikely to result from cortical disinhibition. Collectively, our data demonstrate that knock out of CACN α2δ3 results in some structural abnormalities whose functional implications converge to dedifferentiation of sensory activation.