A new aggressive xenograft model of human colon cancer using cancer-associated fibroblasts.

BACKGROUND: Colorectal cancer is the second leading cause of cancer death. Almost half of the patients present recurrence within 5 years after the treatment of the primary tumor, the majority, with metastasis. On the other hand, in the search for new animal models that simulate metastatic cancer, it has been suggested that fibroblasts immersed in the peritumoral stroma (cancer-associated fibroblasts (CAFs)), play a relevant role in the development of cancer. The objective of this study was to identify an adequate animal model to study metastatic colon cancer and the application of new treatments. METHODS: Human CAFs and normal fibroblasts (NF) for transplant and culture were obtained from surgical fresh samples of patients with adenocarcinoma of sigmoid colon. Stromal cell purity was evaluated by morphology and immunostaining with vimentin (VIM) as a fibroblast marker and anti-proColXIα1 as a specific human CAF marker. Phenotypic characterization of cultured stromal cells was performed by co-staining with mesenchymal and epithelial cell markers. For identification in mice, human CAFs were labeled with the PKH26 red fluorescence dye. Cell line HT-29 was used as tumor cells. Transplant in the head of the pancreas of 34 SCID mice was performed in four different groups, as follows: I. 150,000 CAFS (n = 12), IIa. 1.5 million HT29 cells (n = 7), IIb. 150,000 NF+1.5 million HT29 cells (n = 5), III. 150,000 CAFS+1.5 million HT29 cells (n = 10). After euthanasia performed one month later, histological analysis was made using hematoxylin-eosin and anti-proColXIα1. A histopathological score system based on three features (tumor volume, desmoplasia and number of metastasized organs) was established to compare the tumor severity. RESULTS: The CAFs and NF cultured were proColXIα1+/VIM+, proColXIα1/alphaSMA+ and proColXIα1+/CK19+ in different proportions without differences among them, but the CAFs growth curve was significantly larger than that of the NF (p < 0.05). No tumor developed in those animals that only received CAFs. When comparing group II (a + b) vs. group III, both groups showed 100% hepatic metastases. Median hepatic nodules, tumor burden, lung metastases and severity score were bigger in group III vs group II (a + b), although without being significant, except in the case of the median tumor volume, that was significantly higher in group III (154.8 (76.9-563.2) mm3) vs group II (46.7 (3.7-239.6) mm3), p = 0.04. A correlation was observed between the size of the tumor developed in the pancreas and the metastatic tumor burden in the liver and with the severity score. CONCLUSION: Our experiments demonstrate that cultured CAFs have a higher growth than NF and that when human CAFs are associated to human tumor cells, larger tumors with liver and lung metastases are generated than if only colon cancer cells with/without NF are transplanted. This emphasizes the importance of the tumor stroma, and especially the CAFs, in the development of cancer.

Healthspan and lifespan extension by fecal microbiota transplantation into progeroid mice.

The gut microbiome is emerging as a key regulator of several metabolic, immune and neuroendocrine pathways1,2. Gut microbiome deregulation has been implicated in major conditions such as obesity, type 2 diabetes, cardiovascular disease, non-alcoholic fatty acid liver disease and cancer3-6, but its precise role in aging remains to be elucidated. Here, we find that two different mouse models of progeria are characterized by intestinal dysbiosis with alterations that include an increase in the abundance of Proteobacteria and Cyanobacteria, and a decrease in the abundance of Verrucomicrobia. Consistent with these findings, we found that human progeria patients also display intestinal dysbiosis and that long-lived humans (that is, centenarians) exhibit a substantial increase in Verrucomicrobia and a reduction in Proteobacteria. Fecal microbiota transplantation from wild-type mice enhanced healthspan and lifespan in both progeroid mouse models, and transplantation with the verrucomicrobia Akkermansia muciniphila was sufficient to exert beneficial effects. Moreover, metabolomic analysis of ileal content points to the restoration of secondary bile acids as a possible mechanism for the beneficial effects of reestablishing a healthy microbiome. Our results demonstrate that correction of the accelerated aging-associated intestinal dysbiosis is beneficial, suggesting the existence of a link between aging and the gut microbiota that provides a rationale for microbiome-based interventions against age-related diseases.

The multikinase inhibitor EC-70124 synergistically increased the antitumor activity of doxorubicin in sarcomas.

Cytotoxic drugs like doxorubicin remain as the most utilized agents in sarcoma treatment. However, advanced sarcomas are often resistant, thus stressing the need for new therapies aimed to overcome this resistance. Multikinase inhibitors provide an efficient way to target several pro-tumorigenic pathways using a single agent and may constitute a valuable strategy in the treatment of sarcomas, which frequently show an aberrant activation of pro-tumoral kinases. Therefore, we studied the antitumor activity of EC-70124, an indolocarbazole analog that have demonstrated a robust ability to inhibit a wide range of pro-survival kinases. Evaluation of the phospho-kinase profile in cell-of-origin sarcoma models and/or sarcoma primary cell lines evidenced that PI3K/AKT/mTOR, JAK/STAT or SRC were among the most highly activated pathways. In striking contrast with the structurally related drug midostaurin, EC-70124 efficiently prevented the phosphorylation of these targets and robustly inhibited proliferation through a mechanism associated to the induction of DNA damage, cell cycle arrest and apoptosis. In addition, EC-70124 was able to partially reduce tumor growth in vivo. Importantly, this compound inhibited the expression and activity of ABC efflux pumps involved in drug resistance. In line with this ability, we found that the combined treatment of EC-70124 with doxorubicin resulted in a synergistic cytotoxic effect in vitro and an increased antitumor activity of this cytotoxic drug in vivo. Altogether, these results uncover the capability of the novel multikinase inhibitor EC-70124 to counteract drug resistance in sarcoma and highlight its therapeutic potential when combined with current treatments.

The Chemokine CCL4 (MIP-1ß) Evokes Antinociceptive Effects in Mice: a Role for CD4+ Lymphocytes and Met-Enkephalin.

In the present study, we characterize the antinociceptive effects produced by the chemokine CCL4 in mice. The intraplantar administration of very low doses of CCL4 (0.1-3 pg) produced bilateral antinociception assessed by the unilateral hot-plate test (UHP) without evoking chemotactic responses at the injection site. Moreover, the subcutaneous administration of CCL4 (3-100 pg/kg) also yielded bilateral antinociception in the UHP and the paw pressure test and reduced the number of spinal neurons that express Fos protein in response to noxious stimulation. The implication of peripheral CCR5 but not CCR1 in CCL4-evoked antinociception was deduced from the inhibition produced by systemic but not intrathecal, administration of the CCR5 antagonist DAPTA, and the inefficacy of the CCR1 antagonist J113863. Besides, the inhibition observed after subcutaneous but not intrathecal administration of naloxone demonstrated the involvement of peripheral opioids and the efficacy of naltrindole but not cyprodime or nor-binaltorphimine supported the participation of δ-opioid receptors. In accordance, plasma levels of met-enkephalin, but not ß-endorphin, were augmented in response to CCL4. Likewise, CCL4-evoked antinociception was blocked by the administration of an anti-met-enk antibody. Leukocyte depletion experiments performed with cyclophosphamide, anti-Ly6G, or anti-CD3 antibodies indicated that the antinociceptive effect evoked by CCL4 depends on circulating T lymphocytes. Double immunofluorescence experiments showed a four times more frequent expression of met-enk in CD4+ than in CD8+ T lymphocytes. CCL4-induced antinociception almost disappeared upon CD4+, but not CD8+, lymphocyte depletion with selective antibodies, thus supporting that the release of met-enk from CD4+ lymphocytes underlies the opioid antinociceptive response evoked by CCL4.

Methionine Restriction Extends Lifespan in Progeroid Mice and Alters Lipid and Bile Acid Metabolism.

Dietary intervention constitutes a feasible approach for modulating metabolism and improving the health span and lifespan. Methionine restriction (MR) delays the appearance of age-related diseases and increases longevity in normal mice. However, the effect of MR on premature aging remains to be elucidated. Here, we describe that MR extends lifespan in two different mouse models of Hutchinson-Gilford progeria syndrome (HGPS) by reversing the transcriptome alterations in inflammation and DNA-damage response genes present in this condition. Further, MR improves the lipid profile and changes bile acid levels and conjugation, both in wild-type and in progeroid mice. Notably, treatment with cholic acid improves the health span and lifespan in vivo. These results suggest the existence of a metabolic pathway involved in the longevity extension achieved by MR and support the possibility of dietary interventions for treating progeria.

Trabectedin and Campthotecin Synergistically Eliminate Cancer Stem Cells in Cell-of-Origin Sarcoma Models.

Trabectedin has been approved for second-line treatment of soft tissue sarcomas. However, its efficacy to target sarcoma initiating cells has not been addressed yet. Here, we used pioneer models of myxoid/round cell liposarcoma (MRCLS) and undifferentiated pleomorphic sarcoma (UPS) developed from transformed human mesenchymal stromal/stem cells (MSCs) to evaluate the effect of trabectedin in the cell type responsible for initiating sarcomagenesis and their derived cancer stem cells (CSC) subpopulations. We found that low nanomolar concentrations of trabectedin efficiently inhibited the growth of sarcoma-initiating cells, induced cell cycle arrest, DNA damage and apoptosis. Interestingly, trabectedin treatment repressed the expression of multiple genes responsible for the development of the CSC phenotype, including pluripotency factors, CSC markers and related signaling pathways. Accordingly, trabectedin induced apoptosis and reduced the survival of CSC-enriched tumorsphere cultures with the same efficiency that inhibits the growth of bulk tumor population. In vivo, trabectedin significantly reduced the mitotic index of MRCLS xenografts and inhibited tumor growth at a similar extent to that observed in doxorubicin-treated tumors. Combination of trabectedin with campthotecin (CPT), a chemotherapeutic drug that shows a robust anti-tumor activity when combined with alkylating agents, resulted in a very strong synergistic inhibition of tumor cell growth and highly increased DNA damage and apoptosis induction. Importantly, the enhanced anti-tumor activity of this combination was also observed in CSC subpopulations. These data suggest that trabectedin and CPT combination may constitute a novel strategy to effectively target both the cell-of-origin and CSC subpopulations in sarcoma.

A single-copy Sleeping Beauty transposon mutagenesis screen identifies new PTEN-cooperating tumor suppressor genes.

The overwhelming number of genetic alterations identified through cancer genome sequencing requires complementary approaches to interpret their significance and interactions. Here we developed a novel whole-body insertional mutagenesis screen in mice, which was designed for the discovery of Pten-cooperating tumor suppressors. Toward this aim, we coupled mobilization of a single-copy inactivating Sleeping Beauty transposon to Pten disruption within the same genome. The analysis of 278 transposition-induced prostate, breast and skin tumors detected tissue-specific and shared data sets of known and candidate genes involved in cancer. We validated ZBTB20, CELF2, PARD3, AKAP13 and WAC, which were identified by our screens in multiple cancer types, as new tumor suppressor genes in prostate cancer. We demonstrated their synergy with PTEN in preventing invasion in vitro and confirmed their clinical relevance. Further characterization of Wac in vivo showed obligate haploinsufficiency for this gene (which encodes an autophagy-regulating factor) in a Pten-deficient context. Our study identified complex PTEN-cooperating tumor suppressor networks in different cancer types, with potential clinical implications.

Hyperalgesic and hypoalgesic mechanisms evoked by the acute administration of CCL5 in mice.

We show here that the intraplantar administration of CCL5 in mice produces hyperalgesia at low doses but activates compensatory antinociceptive mechanisms at doses slightly higher. Thus, the injection of 3-10ng of CCL5 evoked thermal hyperalgesia through the activation of CCR1 and CCR5 receptors, as demonstrated by the inhibitory effect exerted by the selective antagonists J113863 (0.01-0.1µg) and DAPTA (0.3-3µg), respectively. The prevention of this hyperalgesia by diclofenac (1-10µg), the inhibitors of COX-1 SC-560 (0.1-1µg) or COX-2 celecoxib (1-5µg), the TRPV1 antagonist capsazepine (0.03-0.3µg) or the TRPA1 antagonist HC030031 (10-50µg) demonstrates the involvement of prostaglandin synthesis and TRP sensitization in CCL5-evoked hyperalgesia. Doses of CCL5 higher than 17µg did not evoke hyperalgesia. However, this effect was restored by the administration of naloxone-methiodide (5µg), nor-binaltorphimine (10mg/kg) or an anti-dynorphin A antibody (0.62-2.5ng). The administration of 30ng of CCL5 also induced hyperalgesia in mice with reduced number of circulating white blood cells in response to cyclophosphamide or with selective neutrophil depletion induced by an anti-Ly6G antibody. In fact, the number of neutrophils present in paws treated with 30ng of CCL5 was greater than in paws receiving the administration of the hyperalgesic dose of 10ng. Finally, the expression of the endogenous opioid peptide dynorphin A was demonstrated by double immunofluorescence assays in these neutrophils attracted by CCL5. These results support previous data describing the hyperalgesic properties of CCL5 and constitute the first indication that a chemokine of the CC group can activate endogenous analgesic mechanisms.

Practical application of stereological methods in experimental kidney animal models. / Aplicación práctica de métodos estereológicos renales en modelos animales experimentales.

The kidneys are vital organs responsible for excretion, fluid and electrolyte balance and hormone production. The nephrons are the kidney's functional and structural units. The number, size and distribution of the nephron components contain relevant information on renal function. Stereology is a branch of morphometry that applies mathematical principles to obtain three-dimensional information from serial, parallel and equidistant two-dimensional microscopic sections. Because of the complexity of stereological studies and the lack of scientific literature on the subject, the aim of this paper is to clearly explain, through animal models, the basic concepts of stereology and how to calculate the main kidney stereological parameters that can be applied in future experimental studies.

Inhibition of SP1 by the mithramycin analog EC-8042 efficiently targets tumor initiating cells in sarcoma.

Tumor initiating cells (TICs), responsible for tumor initiation, and cancer stem cells (CSCs), responsible for tumor expansion and propagation, are often resistant to chemotherapeutic agents. To find therapeutic targets against sarcoma initiating and propagating cells we used models of myxoid liposarcoma (MLS) and undifferentiated pleomorphic sarcoma (UPS) developed from human mesenchymal stromal/stem cells (hMSCs), which constitute the most likely cell-of-origin for sarcoma. We found that SP1-mediated transcription was among the most significantly altered signaling. To inhibit SP1 activity, we used EC-8042, a mithramycin (MTM) analog (mithralog) with enhanced anti-tumor activity and highly improved safety. EC-8042 inhibited the growth of TIC cultures, induced cell cycle arrest and apoptosis and upregulated the adipogenic factor CEBPα. SP1 knockdown was able to mimic the anti-proliferative effects induced by EC-8042. Importantly, EC-8042 was not recognized as a substrate by several ABC efflux pumps involved in drug resistance, and, opposite to the chemotherapeutic drug doxorubicin, repressed the expression of many genes responsible for the TIC/CSC phenotype, including SOX2, C-MYC, NOTCH1 and NFκB1. Accordingly, EC-8042, but not doxorubicin, efficiently reduced the survival of CSC-enriched tumorsphere sarcoma cultures. In vivo, EC-8042 induced a profound inhibition of tumor growth associated to a strong reduction of the mitotic index and the induction of adipogenic differentiation and senescence. Finally, EC-8042 reduced the ability of tumor cells to reinitiate tumor growth. These data suggest that EC-8042 could constitute an effective treatment against both TIC and CSC subpopulations in sarcoma.

Analgesic effects evoked by a CCR2 antagonist or an anti-CCL2 antibody in inflamed mice.

Chemokine CCL2, also known as monocyte chemoattractant protein-1 (MCP-1), is a molecule that in addition to its well-established role in chemotaxis can also act as nociceptor sensitizer. The upregulation of this chemokine in inflamed tissues could suggest its involvement in inflammatory hypernociception. Thus, we have measured CCL2 levels in mice with acute or chronic inflammation due to the intraplantar (i.pl.) injection of carrageenan or complete Freund's adjuvant (CFA), respectively, and we have studied whether inflammatory hyperalgesia or allodynia could be attenuated by blocking CCR2 receptors or neutralizing CCL2 with an anti-CCL2 antibody. A remarkable increase in CCL2 concentration was detected by ELISA in paw homogenates coming from carrageenan- or CFA-inflamed mice, being its expression mainly localized in macrophages, as shown by immunohistochemical assays. The s.c. (0.3-3 mg/kg) or i.pl. (0.3-3 µg) administration of the CCR2 antagonist, RS 504393, dose dependently inhibited thermal hyperalgesia measured in acutely or chronically inflamed mice, whereas s.c. administration of this drug did not reduce inflammatory mechanical allodynia. Furthermore, the inhibition of inflammatory hyperalgesia after the administration of an anti-CCL2 antibody (0.1-1 µg; i.pl.) suggests that CCL2 could be the endogenous chemokine responsible for CCR2-mediated hyperalgesic effects. Besides, the acute administration of the highest antihyperalgesic dose of RS 504393 assayed did not reduce paw tumefaction or modify the presence of inflammatory cells. These results indicate that the blockade of the CCL2/CCR2 system can counteract inflammatory hyperalgesia, being this antinociceptive effect unrelated to a decrease in the inflammatory reaction.

Involvement of CC Chemokine Receptor 1 and CCL3 in Acute and Chronic Inflammatory Pain in Mice.

Chemokines are chemotactic cytokines whose involvement in nociceptive processing is being increasingly recognized. Based on the previous description of the involvement of CC chemokine receptor type 1 (CCR1) in pathological pain, we have assessed the participation of CCR1 and its endogenous ligands CCL3 and CCL5 in hyperalgesia and allodynia in mice after acute inflammation with carrageenan and chronic inflammation with complete Freund's adjuvant (CFA). The subcutaneous administration of the CCR1 antagonist J113863 (3-30 mg/kg; 30 min. before) dose dependently inhibited carrageenan- and CFA-evoked thermal hyperalgesia and mechanical allodynia produced by CFA, but not by carrageenan. The maximal dose of J113863 did not modify the increase in paw thickness induced by carrageenan or CFA. An almost ten times augmentation of CCL3 levels was detected by ELISA assays in both carrageenan and CFA paws, but not in spinal cords of inflamed mice, whereas CCL5 concentrations remained unaltered. Accordingly, a marked increase of CCL3 mRNA expression was observed in inflamed paws, with CCL3 protein detected in neutrophils and macrophages by immunohistochemical experiments. The intraplantar administration of an anti-CCL3 antibody (0.3-3 µg) blocked thermal hyperalgesia in carrageenan- and CFA-inflamed mice as well as CFA-evoked mechanical allodynia. Our data suggest that the increased concentrations of CCL3 present in inflamed tissues can be involved in acute and chronic inflammatory hyperalgesia as well as in chronic mechanical allodynia, and that these hypernociceptive symptoms can be counteracted by its neutralization with an antibody or by the blockade of CCR1 receptors.

Hypernociceptive responses following the intratibial inoculation of RM1 prostate cancer cells in mice.

BACKGROUND: Pain due to bone metastases of prostatic origin is a relevant clinical issue. We study here the nociceptive responses obtained in mice receiving the intratibial inoculation of RM1 prostate cancer cells. METHODS: 10(2) -10(5) RM1 cells were inoculated to C57BL/6 mice and tumor development was analysed histologically and with luciferase-expressing RM1 cells. Spinal astroglial (GFAP) or microglial (Iba-1) expression was assessed with immunohistochemical methods and hypernociception was measured by the unilateral hot plate, the paw pressure and the von Frey tests. The analgesic effect of morphine, zoledronic acid or the CCR2 antagonist RS504393 was measured. Levels of the chemokines CCL2, CCL3, and CCL5 were determined by ELISA. RESULTS: The inoculation of 10(3) RM1 cells induced tumoral growth in bone with a mixed osteoclastic/osteoblastic pattern and evoked astroglial, but not microglial, activation in the spinal cord. Hyperalgesia and allodynia were already established four days after inoculation and dose-dependently inhibited by the s.c. administration of morphine (1-5 mg/kg) or zoledronic acid (1-3 mg/kg). CCL2 and CCL5, but not CCL3, were released by RM1 cells in culture whereas only an increased presence of CCL2 was found in bone tumor homogenates. The administration of the CCR2 antagonist RS504393 (0.3-3 mg/kg) inhibited RM1 induced thermal hyperalgesia without modifying mechanical allodynia. CONCLUSION: The intratibial inoculation of RM1 cells in immunocompetent mice induces hypernociceptive responses and can be useful to perform studies of bone cancer induced pain related to androgen-independent prostate cancer. The antinociceptive role derived from the blockade of the CCR2 chemokine receptors is further envisaged.

[Cystic hamartoma of the renal pelvis: unfrequent finding]. / Hamartoma quístico de pelvis renal, un hallazgo infrecuente.

OBJECTIVES: This cystic hamartoma of the renal pelvis is a very unfrequent benign tumor. We report one case emphasizing its histology and performed a bibliographic review. METHODS: Mid age female patient with the incidental diagnosis of a renal mass taking up the pelvis. RESULTS: A radical nephrectomy was performed with the pathologic report of cystic hamartoma of the renal pelvis. CONCLUSIONS: It is a cystic renal tumor with well-defined histologic and immunohistochemical criteria, and it probably does not involve any danger for the patient's live.

[Bladder metastasis of renal carcinoma]. / Metástasis vesical de carcinoma renal.

OBJECTIVES: We report the case of a patient who had underwent surgery for a clear cell renal carcinoma 2 years before presenting with metastasic extension to bladder on follow-up. METHODS: Radiological finding of a bladder mass during follow-up after nephrectomy. RESULTS: TURBT was carried out with the pathologic report of clear cell carcinoma, compatible with a primary renal origin. CONCLUSIONS: Bladder is a very rare place for metastasis from kidney tumors. Prognosis will depend on the time of appearance of such metastases.

[Obstructive urethral angioleiomyoma]. / Angioleiomioma uretral obstructivo.

OBJECTIVES: To report one case of urethral angioleiomyoma because of the rare site, and to review its main characteristics. METHODS: We report one case of male urethra angioleiomyoma presenting with obstructive symptoms. RESULTS: We performed excision and end to end anastomosis of a small bulbar urethra stenosis. Pathology reported angioleiomyoma. CONCLUSIONS: Angioleiomyoma is a benign tumor arising from smooth muscle within blood vessel walls. It is extremely rare in the urethra. It is more common in females. Recurrence and metastasis are exceptional. It shows scarce or moderate cellularity with predominance of fusiform cells. They may express smooth muscle vimentin, desmin, and actin. The best therapeutic option is resection.