Serum Symmetric Dimethylarginine Concentration in Greyhound Pups and Adults.

Greyhounds have developed numerous physiologic and hematologic adaptations specific to the breed. Adult Greyhounds have significantly higher serum creatinine (sCr) concentrations than non-Greyhound dogs; this is primarily attributed to their large muscle mass. Adult Greyhounds also have significantly higher symmetric dimethylarginine (SDMA) concentrations than non-Greyhound dogs. In this study we evaluated serum SDMA and sCr concentrations in 48 Greyhound pups from 11 litters, with no exposure to racing, in order to determine if the higher SDMA or sCr concentration in adults is a breed-related trait or is associated with extra-physiological causes (e.g., racing environment, drug administration, etc.). The distribution of SDMA concentration for all the pups (mean: 0.70 µmol/L; 95% CI: 0.70-0.74 µmol/L) was not statistically different from that found in a group of 183 adult Greyhounds; whereas, distribution of sCr concentration in the pups (mean: 76.9 µmol/L; 95% CI: 70.7-83.1 µmol/L) was significantly different than that observed in adult Greyhounds, suggesting that the 2 samples were drawn from different populations. This study provides evidence to support that the higher serum SDMA concentration in Greyhounds is a breed-related trait.

Effect of packed red blood cell transfusion on thromboelastographic tracings in dogs with naturally occurring anemia.

OBJECTIVE: To assess the effect of packed RBC (pRBC) transfusion on thromboelastographic (TEG) tracings in dogs with naturally occurring anemia. ANIMALS: 22 clinically anemic dogs that received a pRBC transfusion. PROCEDURES: For each dog, a blood sample was collected before and within 3 hours after completion of the pRBC transfusion for a CBC, nonactivated TEG analysis, and measurement of blood viscosity. Wilcoxon signed rank tests were used to compare CBC, viscosity, and TEG variables between pretransfusion and posttransfusion blood samples. Multivariable linear regression was used to assess the effects of pretransfusion-posttransfusion changes in Hct, WBC count, and platelet count on changes in TEG variables. RESULTS: Median posttransfusion Hct (21%; range, 13% to 34%) was significantly greater than the median pretransfusion Hct (12.5%; range, 7% to 29%). Packed RBC transfusion was associated with a median increase in Hct of 6.2% (range, 1.2% to 13%). Maximum amplitude significantly decreased from 74.9 to 73.8 mm and clot strength significantly decreased from 14,906 to 14,119 dynes/s after pRBC transfusion. Blood viscosity significantly increased, whereas platelet and WBC counts significantly decreased after transfusion. Multivariable linear regression revealed that pretransfusion-posttransfusion changes in Hct, WBC count, and platelet count were not associated with changes in TEG variables. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that pRBC transfusion had only small effects on the TEG tracings of hemodynamically stable dogs. Therefore, large changes in TEG tracings following pRBC transfusion are unlikely to be the result of the transfusion and should be investigated further.

Outcome and treatments of dogs with aortic thrombosis: 100 cases (1997-2014).

BACKGROUND: Aortic thrombosis (ATh) is an uncommon condition in dogs, with limited understanding of risks factors, outcomes, and treatments. OBJECTIVES/HYPOTHESIS: To describe potential risk factors, outcome, and treatments in dogs with ATh. ANIMALS: Client-owned dogs with a diagnosis of ATh based on ultrasonographic or gross necropsy examination. METHOD: Multicentric retrospective study from 2 academic institutions. RESULTS: One hundred dogs were identified. Anti-thrombin diagnosis, 35/100 dogs were nonambulatory. The dogs were classified as acute (n = 27), chronic (n = 72), or unknown (n = 1). Fifty-four dogs had at least one comorbidity thought to predispose to ATh, and 23 others had multiple comorbidities. The remaining 23 dogs with no obvious comorbidities were classified as cryptogenic. Concurrent illnesses potentially related to the development of ATh included protein-losing nephropathy (PLN) (n = 32), neoplasia (n = 22), exogenous corticosteroid administration (n = 16), endocrine disease (n = 13), and infection (n = 9). Dogs with PLN had lower antithrombin activity than those without PLN (64% and 82%, respectively) (P = .04). Sixty-five dogs were hospitalized with 41 subsequently discharged. Sixteen were treated as outpatient and 19 euthanized at admission. In-hospital treatments varied, but included thrombolytics (n = 12), alone or in combination with thrombectomy (n = 9). Fifty-seven dogs survived to discharge. Sixteen were alive at 180 days. Using regression analysis, ambulation status at the time of presentation was significantly correlated with survival-to-discharge (P < .001). CONCLUSIONS/CLINICAL IMPORTANCE: Dogs with ATh have a poor prognosis, with nonambulatory dogs at the time of presentation having worse outcome. Although the presence of comorbid conditions associated with hypercoagulability is common, an underlying cause for ATh was not always identified.

Hyperkalemia during general anesthesia in two Greyhounds.

CASE DESCRIPTION: A 36-kg (79-lb) castrated male Greyhound (dog 1) and a 25-kg (55 lb) spayed female Greyhound (dog 2) underwent general anesthesia for dental care with similar perianesthetic protocols on multiple occasions from 2013 to 2016. Both dogs had periodontal disease but were otherwise deemed healthy. Both dogs developed clinically relevant hyperkalemia, with signs including loss of P waves on ECG tracings, during multiple anesthetic events. CLINICAL FINDINGS: Dog 1 developed hyperkalemia during 2 of 2 anesthetic events, with ECG changes noted during the first event. Dog 2 developed hyperkalemia during 3 of 4 anesthetic events, with ECG changes identified during the second and third events. Serum potassium concentration for both dogs was within the reference range prior to and between anesthetic events. No underlying etiopathogenesis for hyperkalemia was identified for either dog. TREATMENT AND OUTCOME: In each hyperkalemic event, the clinician stopped the dental procedure and continued to provide supportive care and monitoring while the dog recovered from anesthesia. The ECG changes resolved, and serum potassium concentration returned to the reference range rapidly after inhalant anesthetic administration was discontinued. The dogs were discharged from the hospital without further complications. CLINICAL RELEVANCE: Hyperkalemia in anesthetized Greyhounds resulted in serious cardiac conduction abnormalities, which could be potentially fatal if not recognized and promptly treated. Further investigation into the etiopathogenesis, prevention and treatment strategies, and genetic or familial components of this condition is indicated.

Risk-modeling of dog osteosarcoma genome scans shows individuals with Mendelian-level polygenic risk are common.

BACKGROUND: Despite the tremendous therapeutic advances that have stemmed from somatic oncogenetics, survival of some cancers has not improved in 50 years. Osteosarcoma still has a 5-year survival rate of 66%. We propose the natural canine osteosarcoma model can change that: it is extremely similar to the human condition, except for being highly heritable and having a dramatically higher incidence. Here we reanalyze published genome scans of osteosarcoma in three frequently-affected dog breeds and report entirely new understandings with immediate translational indications. RESULTS: First, meta-analysis revealed association near FGF9, which has strong biological and therapeutic relevance. Secondly, risk-modeling by multiple logistic regression shows 22 of the 34 associated loci contribute to risk and eight have large effect sizes. We validated the Greyhound stepwise model in our own, independent, case-control cohort. Lastly, we updated the gene annotation from approximately 50 genes to 175, and prioritized those using cross-species genomics data. Mostly positional evidence suggests 13 genes are likely to be associated with mapped risk (including MTMR9, EWSR1 retrogene, TANGO2 and FGF9). Previous annotation included seven of those 13 and prioritized four by pathway enrichment. Ten of our 13 priority genes are in loci that contribute to risk modeling and thus can be studied epidemiologically and translationally in pet dogs. Other new candidates include MYCN, SVIL and MIR100HG. CONCLUSIONS: Polygenic osteosarcoma-risk commonly rises to Mendelian-levels in some dog breeds. This justifies caninized animal models and targeted clinical trials in pet dogs (e.g., using CDK4/6 and FGFR1/2 inhibitors).

SETD2 Is Recurrently Mutated in Whole-Exome Sequenced Canine Osteosarcoma.

Osteosarcoma is a debilitating bone cancer that affects humans, especially children and adolescents. A homologous form of osteosarcoma spontaneously occurs in dogs, and its differential incidence observed across breeds allows for the investigation of tumor mutations in the context of multiple genetic backgrounds. Using whole-exome sequencing and dogs from three susceptible breeds (22 golden retrievers, 21 Rottweilers, and 23 greyhounds), we found that osteosarcoma tumors show a high frequency of somatic copy-number alterations (SCNA), affecting key oncogenes and tumor-suppressor genes. The across-breed results are similar to what has been observed for human osteosarcoma, but the disease frequency and somatic mutation counts vary in the three breeds. For all breeds, three mutational signatures (one of which has not been previously reported) and 11 significantly mutated genes were identified. TP53 was the most frequently altered gene (83% of dogs have either mutations or SCNA in TP53), recapitulating observations in human osteosarcoma. The second most frequently mutated gene, histone methyltransferase SETD2, has known roles in multiple cancers, but has not previously been strongly implicated in osteosarcoma. This study points to the likely importance of histone modifications in osteosarcoma and highlights the strong genetic similarities between human and dog osteosarcoma, suggesting that canine osteosarcoma may serve as an excellent model for developing treatment strategies in both species.Significance: Canine osteosarcoma genomics identify SETD2 as a possible oncogenic driver of osteosarcoma, and findings establish the canine model as a useful comparative model for the corresponding human disease. Cancer Res; 78(13); 3421-31. ©2018 AACR.

Comparative genomics of canine hemoglobin genes reveals primacy of beta subunit delta in adult carnivores.

BACKGROUND: The main function of hemoglobin (Hb) is to transport oxygen in the circulation. It is among the most highly studied proteins due to its roles in physiology and disease, and most of our understanding derives from comparative research. There is great diversity in Hb gene evolution in placental mammals, mostly in the repertoire and regulation of the ß-globin subunits. Dogs are an ideal model in which to study Hb genes because: 1) they are members of Laurasiatheria, our closest relatives outside of Euarchontoglires (including primates, rodents and rabbits), 2) dog breeds are isolated populations with their own Hb-associated genetics and diseases, and 3) their high level of health care allows for development of biomedical investigation and translation. RESULTS: We established that dogs have a complement of five α and five ß-globin genes, all of which can be detected as spliced mRNA in adults. Strikingly, HBD, the allegedly-unnecessary adult ß-globin protein in humans, is the primary adult ß-globin in dogs and other carnivores; moreover, dogs have two active copies of the HBD gene. In contrast, the dominant adult ß-globin of humans, HBB, has high sequence divergence and is expressed at markedly lower levels in dogs. We also showed that canine HBD and HBB genes are complex chimeras that resulted from multiple gene conversion events between them. Lastly, we showed that the strongest signal of evolutionary selection in a high-altitude breed, the Bernese Mountain Dog, lies in a haplotype block that spans the ß-globin locus. CONCLUSIONS: We report the first molecular genetic characterization of Hb genes in dogs. We found important distinctions between adult ß-globin expression in carnivores compared to other members of Laurasiatheria. Our findings are also likely to raise new questions about the significance of human HBD. The comparative genomics of dog hemoglobin genes sets the stage for diverse research and translation.

Effect of ascorbic acid on storage of Greyhound erythrocytes.

OBJECTIVE: To assess changes in biochemical and biophysical properties of canine RBCs during cold (1° to 6°C) storage in a licensed RBC additive solution (the RBC preservation solution designated AS-1) supplemented with ascorbic acid. SAMPLE: Blood samples from 7 neutered male Greyhounds; all dogs had negative results when tested for dog erythrocyte antigen 1.1. PROCEDURES: Blood was collected into citrate-phosphate-dextrose and stored in AS-1. Stored RBCs were supplemented with 7.1mM ascorbic acid or with saline (0.9% NaCl) solution (control samples). Several biochemical and biophysical properties of RBCs were measured, including percentage hemolysis, oxygen-hemoglobin equilibrium, and the kinetic rate constants for O2 dissociation, carbon monoxide association, and nitric oxide dioxygenation. RESULTS: Greyhound RBCs stored in AS-1 supplemented with ascorbic acid did not have significantly decreased hemolysis, compared with results for the control samples, during the storage period. CONCLUSIONS AND CLINICAL RELEVANCE: In this study, ascorbic acid did not reduce hemolysis during storage. Several changes in stored canine RBCs were identified as part of the hypothermic storage lesion.

Plasma N-terminal pro-B-type natriuretic peptide concentration in healthy retired racing Greyhounds.

BACKGROUND: N-terminal pro-B-type natriuretic peptide (NT-proBNP) is a cardiac biomarker whose plasma concentration is high in some dogs with cardiopulmonary disease. NT-proBNP is a diagnostic tool that can be used to help determine if a patient has congestive heart failure. Greyhounds have functional heart murmurs, relative cardiomegaly, and high serum cTnI concentration. OBJECTIVES: The purpose of the study was to evaluate the plasma concentration of NT-proBNP in healthy Greyhounds and compare it to non-Greyhound dogs. METHODS: We prospectively evaluated healthy client-owned dogs including retired racing Greyhounds and non-Greyhounds. Plasma was obtained and transferred into tubes containing a protease inhibitor and submitted for a specific NT-proBNP ELISA assay. RESULTS: The plasma NT-proBNP concentration in Greyhounds was significantly higher than in non-Greyhound control dogs (946 vs 632 pmol/L; P < .005); 46% of Greyhounds had NT-proBNP > 1000 pmol/L. CONCLUSIONS: Plasma NT-proBNP concentration in Greyhounds is high and should be interpreted with caution.

Comparison of high-dose intermittent and low-dose continuous oral artemisinin in dogs with naturally occurring tumors.

To evaluate the clinical toxicity and activity of orally administered artemisinin in dogs with spontaneous tumors, 24 client-owned dogs were randomly divided into two groups and received either low-continuous dose (3 mg/kg q 24 hr) or high-dose intermittent (three doses of 45 mg/kg q 6 hr repeated q 1 wk) of artemisinin per os. Treatment was continued for 21 days. Dogs were evaluated weekly for clinical effect and at the end of the treatment for hematologic and biochemical adverse events. Whole blood concentrations of artemisinin and dihydroartemisinin were measured by liquid chromatography/tandem mass spectrometry after the first dose of artemisinin in three dogs in each group. Blood concentrations of artemisinin and dihydroartemisinin were <0.1 µM at all time points, and there was no difference in blood concentration between the two dosing groups. The most frequent adverse event was anorexia, which was observed in 11% of the low-dose group and 29% of the high-dose group. Oral artemisinin, both in low-dose continuous and high-dose intermittent, is well tolerated in dogs but results in low bioavailability. Parenteral administration should be considered for future studies.

Effects of hematocrit and red blood cell-independent viscosity on canine thromboelastographic tracings.

BACKGROUND: It is well established that hematocrit (Hct) influences whole blood thromboelastography (TEG) tracings. Previous studies showed hypercoagulable TEG tracings in anemic patients despite clinical expectations that anemia often prolongs bleeding. TEG is a viscoelastic assessment of clot kinetics, and Hct is the main determinant of whole blood viscosity. TEG changes in anemia may be an in vitro artifact due to Hct effect on blood viscosity rather than true in vivo changes in hemostasis. The effect of changes in whole blood viscosity on TEG independent of Hct is not well understood. STUDY DESIGN AND METHODS: Twenty-one blood samples from seven dogs were manipulated to produce one of three Hct conditions (45, 20, and 10%). Each was tested in two situations: viscosity adjusted to normal by adding alginate (ALG) or dilution with equal volume of saline (SAL). Both samples were analyzed with TEG simultaneously. RESULTS: Twenty percent Hct plus ALG and 10% Hct plus ALG were significantly more viscous than their SAL counterparts (p=0.0156). Ten percent Hct plus SAL, 20% Hct plus SAL, and 45% Hct plus SAL all had different viscosities (p=0.006). Twenty percent Hct plus SAL and 10% Hct plus SAL had significantly shorter K and higher angle, MA, and G compared to their ALG counterparts as well as 45% Hct plus SAL (p<0.05). CONCLUSIONS: ALG samples with low Hct, normal viscosity showed hypocoagulable tracings, whereas SAL samples with low Hct, low viscosity showed hypercoagulable tracings. TEG variables are influenced by whole blood viscosity altered with ALG, independently of Hct.

Effect of sevoflurane anesthesia and blood donation on the sonographic appearance of the spleen in 60 healthy cats.

The aims of this prospective study were to describe the normal sonographic size and appearance of the feline spleen, and to test effects of sevoflurane anesthesia and blood collection on sonographic characteristics. Sonographic evaluations were performed in 60 healthy blood donor cats prior to anesthesia and blood collection (baseline), after anesthesia induction, after completion of blood collection while under anesthesia, and 7-10 days after blood collection. Transverse sonographic images of the splenic mid-body were made. Splenic height, width, cross-sectional area, echogenicity relative to the left renal cortex, and echotexture were recorded. Height, width, and cross-sectional area were compared across time periods using linear-mixed effects models. Holm's method was applied to adjust for multiplicity and control the overall Type 1 error rate at α = 0.05. Normal transverse sonographic splenic measurements (mean ± standard deviation) at baseline were: height 8.2 ± 1.4 mm; width 26.7 ± 4.4 mm; and CSA 1.6 ± 0.5 cm(2) . While there were statistically significant differences in splenic height, width, and cross-sectional area after sevoflurane anesthesia and/or blood collection, differences were of low magnitude. The spleen was diffusely homogenous and subjectively isoechoic or mildly hyperechoic relative to the left renal cortex. In 77% of castrated male cats, the spleen was isoechoic to the left renal cortex. We conclude that sevoflurane anesthesia and blood donation do not subjectively alter splenic dimensions, echogenicity and echotexture in normal cats.

Epsilon aminocaproic acid for the prevention of delayed postoperative bleeding in retired racing greyhounds undergoing gonadectomy.

OBJECTIVE: To evaluate the effects of epsilon aminocaproic acid (EACA) on the prevalence of postoperative bleeding in retired racing Greyhounds (RRG), and to assess its effects on selected thrombelastography (TEG) and fibrinolysis variables. STUDY DESIGN: Double-blinded, prospective, randomized study. METHODS: 100 RRG had elective ovariohysterectomy or orchiectomy and were administered EACA or placebo for 3 days after surgery. TEG variables were analyzed preoperatively and 24, 48, and 72 hours after surgery. RESULTS: Thirty percent (15/50) of RRG in the placebo group had delayed postoperative bleeding starting 36-48 hours after surgery compared with 10% (5/50) in the EACA group (P = .012). On the TEG variables, the slopes for R and K time were significantly different between treatment groups (P <.05); the R and K time decreased over time in the EACA group after surgery whereas they increased in the placebo group. The angle, maximal amplitude (MA), and G slopes were also significantly different between treatment groups (P = .001, .001, and .006, respectively). The angle, MA, and G increased postoperatively over time in the EACA group and decreased in the placebo group. All these changes are supportive of hypercoagulability associated with EACA administration. CONCLUSION: Postoperative administration of EACA significantly decreased the prevalence of postoperative bleeding in RRG undergoing surgery by increasing the clot strength.

Retrospective evaluation of the effectiveness of epsilon aminocaproic acid for the prevention of postamputation bleeding in retired racing Greyhounds with appendicular bone tumors: 46 cases (2003-2008).

OBJECTIVES: To determine the frequency of delayed postoperative bleeding in retired racing Greyhounds with appendicular bone tumors undergoing limb amputations. To identify if administration of epsilon-aminocaproic acid (EACA) was effective on the prevention of postoperative bleeding. DESIGN: Retrospective study from December 2003 to December 2008. SETTING: Veterinary university teaching hospital. ANIMALS: Forty-six retired racing Greyhounds (RRGs) diagnosed with primary appendicular bone tumors that underwent limb amputation were included in the study. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Thirteen of 46 RRGs (28%) included in the study had delayed postoperative bleeding starting 48-72 h after surgery. Bleeding episodes included cutaneous, subcutaneous, and external bleeding that extended from the area of the surgical site that became widespread within hours, and that required administration of blood components. A paired t-test suggests that there was a significant decrease in PCV postoperatively for both dogs that bled and dogs that did not bleed (P < 0.0001). Forty of 46 RRGs (86%) received either fresh frozen plasma (FFP) or EACA or both, for the prevention of postoperative bleeding. A logistic regression model determined that dogs that did not receive EACA were 5.7 times more likely to bleed than dogs that did receive EACA, when controlling for whether or not they received FFP (95% CI: 1.02-32.15, P = 0.047). CONCLUSION: This retrospective study suggests that preemptive postoperative administration of EACA appears to be efficacious in decreasing the frequency of bleeding in RRGs undergoing limb amputation; however, a prospective study is warranted to corroborate its effectiveness.

Seroprevalence of Ehrlichia canis, Ehrlichia chaffeensis and Ehrlichia ewingii in dogs in North America.

BACKGROUND: This study evaluated the exposure of dogs to three different Ehrlichia spp. in the south and central regions of the United States where vector-borne disease prevalence has been previously difficult to ascertain, particularly beyond the metropolitan areas. METHODS: Dog blood samples (n = 8,662) were submitted from 14 veterinary colleges, 6 private veterinary practices and 4 diagnostic laboratories across this region. Samples were tested for E. canis, E. chaffeensis and E. ewingii specific antibodies using peptide microtiter ELISAs. RESULTS: Overall, E. canis, E. chaffeensis and E. ewingii seroprevalence was 0.8%, 2.8%, and 5.1%, respectively. The highest E. canis seroprevalence (2.3%) was found in a region encompassing Arkansas, Louisiana, Oklahoma, Tennessee and Texas. E. chaffeensis seroreactivity was 6.6% in the central region (Arkansas, Kansas, Missouri, and Oklahoma) and 4.6% in the southeast region (Georgia, Maryland, North Carolina, South Carolina, Tennessee and Virginia). Seroreactivity to E. ewingii was also highest in the central region (14.6%) followed by the southeast region (5.9%). The geospatial pattern derived from E. chaffeensis and E. ewingii seropositive samples was similar to previous reports based on E. chaffeensis seroreactivity in white-tailed deer and the distribution of human monocytic ehrlichiosis (HME) cases reported by the CDC. CONCLUSIONS: The results of this study provide the first large scale regional documentation of exposure to E. canis, E. chaffeensis and E. ewingii in pet dogs, highlighting regional differences in seroprevalence and providing the basis for heightened awareness of these emerging vector-borne pathogens by veterinarians and public health agencies.

Structure of Greyhound hemoglobin: origin of high oxygen affinity.

This study presents the crystal structure of Greyhound hemoglobin (GrHb) determined to 1.9 Šresolution. GrHb was found to crystallize with an α1ß1 dimer in the asymmetric unit and belongs to the R2 state. Oxygen-affinity measurements combined with the fact that GrHb crystallizes in the R2 state despite the high-salt conditions used for crystallization strongly indicate that GrHb can serve as a model high-oxygen-affinity hemoglobin (Hb) for higher mammals, especially humans. Structural analysis of GrHb and its comparison with the R2-state of human Hb revealed several regions that can potentially contribute to the high oxygen affinity of GrHb and serve to rationalize the additional stability of the R2-state of GrHb. A previously well studied hydrophobic cluster of bar-headed goose Hb near α119 was also incorporated in the comparison between GrHb and human Hb. Finally, a structural comparison with generic dog Hb and maned wolf Hb was conducted, revealing that in contrast to GrHb these structures belong to the R state of Hb and raising the intriguing possibility of an additional allosteric factor co-purifying with GrHb that can modulate its quaternary structure.

Blood gas analysis and cooximetry in retired racing Greyhounds.

OBJECTIVE: The purposes of this study were to evaluate the oxygen affinity of hemoglobin (Hb) in healthy retired racing Greyhounds via cooximetry, and to establish reference intervals for blood gases and cooximetry in this breed. DESIGN: Prospective clinical study. SETTING: University Teaching Hospital. ANIMALS: Fifty-seven Greyhounds and 30 non-Greyhound dogs. INTERVENTIONS: Venous blood samples were collected from the jugular vein and placed into heparinized tubes. The samples were analyzed within 30 minutes of collection using a blood gas analyzer equipped with a cooximeter. MEASUREMENTS AND MAIN RESULTS: Greyhounds had significantly higher pH, PO(2) , oxygen saturation, oxyhemoglobin, total Hb, oxygen content, and oxygen capacity and significantly lower deoxyhemoglobin and P(50) when compared with non-Greyhound dogs. CONCLUSION: These findings support the fact that this breed is able to carry a higher concentration of total oxygen in the blood. As reported previously, this breed also has lower P(50) and, therefore, high oxygen affinity. In light of recent findings suggesting that in certain tissues a high affinity for oxygen is beneficial, this adaptation may be of benefit during strenuous exercise.

Hydrophilic agarose macrobead cultures select for outgrowth of carcinoma cell populations that can restrict tumor growth.

Cancer cells and their associated tumors have long been considered to exhibit unregulated proliferation or growth. However, a substantial body of evidence indicates that tumor growth is subject to both positive and negative regulatory controls. Here, we describe a novel property of tumor growth regulation that is neither species nor tumor-type specific. This property, functionally a type of feedback control, is triggered by the encapsulation of neoplastic cells in a growth-restricting hydrogel composed of an agarose matrix with a second coating of agarose to form 6- to 8-mm diameter macrobeads. In a mouse cell model of renal adenocarcinoma (RENCA cells), this process resulted in selection for a stem cell-like subpopulation which together with at least one other cell subpopulation drove colony formation in the macrobeads. Cells in these colonies produced diffusible substances that markedly inhibited in vitro and in vivo proliferation of epithelial-derived tumor cells outside the macrobeads. RENCA cells in monolayer culture that were exposed to RENCA macrobead-conditioned media exhibited cell-cycle accumulation in S phase due to activation of a G(2)/M checkpoint. At least 10 proteins with known tumor suppression functions were identified by analysis of RENCA macrobead-conditioned media, the properties of which offer opportunities to further dissect the molecular basis for tumor growth control. More generally, macrobead culture may permit the isolation of cancer stem cells and other cells of the stem cell niche, perhaps providing strategies to define more effective biologically based clinical approaches to treat neoplastic disease.