Tumor-localized interleukin-2 and interleukin-12 combine with radiation therapy to safely potentiate regression of advanced malignant melanoma in pet dogs.

PURPOSE: Interleukin-2 and -12 cytokines have potent anti-cancer activity, but suffer a narrow therapeutic window due to off-tumor immune cell activation. Engineering cytokines with the ability to bind and associate with tumor collagen after intratumoral injection potentiated response without toxicity in mice, and was previously safe in pet dogs with sarcoma. Here we sought to test the efficacy of this approach with in dogs with advanced melanoma. EXPERIMENTAL DESIGN: This study examined fifteen client-owned dogs with histologically- or cytologically-confirmed malignant melanoma who received a single 9 Gray fraction of radiation therapy, followed by six cycles of combined collagen-anchored IL-2 and IL-12 therapy Q2W. Cytokine dosing followed a 3+3 dose escalation design, with the initial cytokine dose chosen from prior evaluation in canine sarcomas. No exclusion criteria for tumor stage or metastatic burden, age, weight, or neuter status were applied for this trial. RESULTS: Median survival regardless of tumor stage or dose level was 256 days and 10/13 (76.9%) dogs that completed treatment had CT-measured tumor regression at the treated lesion. In dogs with metastatic disease, 8/13 (61.5%) dogs had partial responses across their combined lesions, evidence of locoregional response. Profiling by Nanostring of treatment-resistant dogs revealed that B2m loss was predictive of poor response to this therapy. CONCLUSIONS: Collectively, these results confirm the ability of locally administered tumor-anchored cytokines to potentiate responses at regional disease sites when combined with radiation. This evidence supports the clinical translation of this approach and highlights the utility of comparative investigation in canine cancers.

Minimizing higher-order aggregation maximizes iron mobilization by small molecules.

The natural product hinokitiol mobilizes iron across lipid bilayers at low concentrations and restores hemoglobinization in iron transporter protein-deficient systems. But hinokitiol fails to similarly mobilize iron at higher concentrations, limiting its uses in chemical biology and medicine. Here we show that at higher concentrations, hinokitiol3:Fe(III) complexes form large, higher-order aggregates, leading to loss of transmembrane iron mobilization. Guided by this understanding and systematic structure-function studies enabled by modular synthesis, we identified FeM-1269, which minimally aggregates and dose-dependently mobilizes iron across lipid bilayers even at very high concentrations. In contrast to hinokitiol, FeM-1269 is also well-tolerated in animals at high doses for extended periods of time. In a mouse model of anemia of inflammation, FeM-1269 increases serum iron, transferrin saturation, hemoglobin and hematocrit. This rationally developed iron-mobilizing small molecule has enhanced potential as a molecular prosthetic for understanding and potentially treating iron transporter deficiencies.

Alternating rabacfosadine and doxorubicin for treatment of naïve canine lymphoma.

The current standard of care treatment for canine lymphoma is a multi-agent, CHOP-based chemotherapy protocol. Single agent doxorubicin (DOX) is less burdensome; however, multi-agent chemotherapy protocols are often superior. The recently approved drug rabacfosadine (RAB, Tanovea) provides an attractive option for combination therapy with DOX, as both drugs demonstrate efficacy against lymphoma and possess different mechanisms of action. A previous study evaluating alternating RAB/DOX reported an overall response rate (ORR) of 84%, with a median progression-free survival time (PFS) of 194 days. The aim of this prospective trial was to evaluate the same protocol in an additional population of dogs. Fifty-nine dogs with treatment naïve lymphoma were enrolled. RAB (1.0 mg/kg IV) was alternated with DOX (30 mg/m2 IV) every 21 days for up to six total treatments (3 cycles). Response assessment and adverse event (AE) evaluation were performed every 21 days using VCOG criteria. The ORR was 93% (79% CR, 14% PR). The median time to maximal response was 21.5 days; median PFS was 199 days. T cell immunophenotype and lack of treatment response were predictive of inferior outcomes. AEs were mostly gastrointestinal. Six dogs developed presumed or confirmed pulmonary fibrosis; four were grade 5. One dog experienced grade 3 extravasation injury with RAB that resolved with supportive treatment. These data mirror those of the previously reported RAB/DOX study, and support the finding that alternating RAB/DOX is a reasonable treatment option for canine lymphoma.

Tumor-localized interleukin-2 and interleukin-12 combine with radiation therapy to safely potentiate regression of advanced malignant melanoma in pet dogs.

The clinical use of interleukin-2 and -12 cytokines against cancer is limited by their narrow therapeutic windows due to on-target, off-tumor activation of immune cells when delivered systemically. Engineering IL-2 and IL-12 to bind to extracellular matrix collagen allows these cytokines to be retained within tumors after intralesional injection, overcoming these clinical safety challenges. While this approach has potentiated responses in syngeneic mouse tumors without toxicity, the complex tumor-immune interactions in human cancers are difficult to recapitulate in mouse models of cancer. This has driven an increased role for comparative oncology clinical trials in companion (pet) dogs with spontaneous cancers that feature analogous tumor and immune biology to human cancers. Here, we report the results from a dose-escalation clinical trial of intratumoral collagen-binding IL-2 and IL-12 cytokines in pet dogs with malignant melanoma, observing encouraging local and regional responses to therapy that may suggest human clinical benefit with this approach.

Tuning sterol extraction kinetics yields a renal-sparing polyene antifungal.

Decades of previous efforts to develop renal-sparing polyene antifungals were misguided by the classic membrane permeabilization model1. Recently, the clinically vital but also highly renal-toxic small-molecule natural product amphotericin B was instead found to kill fungi primarily by forming extramembraneous sponge-like aggregates that extract ergosterol from lipid bilayers2-6. Here we show that rapid and selective extraction of fungal ergosterol can yield potent and renal-sparing polyene antifungals. Cholesterol extraction was found to drive the toxicity of amphotericin B to human renal cells. Our examination of high-resolution structures of amphotericin B sponges in sterol-free and sterol-bound states guided us to a promising structural derivative that does not bind cholesterol and is thus renal sparing. This derivative was also less potent because it extracts ergosterol more slowly. Selective acceleration of ergosterol extraction with a second structural modification yielded a new polyene, AM-2-19, that is renal sparing in mice and primary human renal cells, potent against hundreds of pathogenic fungal strains, resistance evasive following serial passage in vitro and highly efficacious in animal models of invasive fungal infections. Thus, rational tuning of the dynamics of interactions between small molecules may lead to better treatments for fungal infections that still kill millions of people annually7,8 and potentially other resistance-evasive antimicrobials, including those that have recently been shown to operate through supramolecular structures that target specific lipids9.

The combination of PAC-1 and entrectinib for the treatment of metastatic uveal melanoma.

The treatment of metastatic uveal melanoma remains a major clinical challenge. Procaspase-3, a proapoptotic protein and precursor to the key apoptotic executioner caspase-3, is overexpressed in a wide range of malignancies, and the drug PAC-1 leverages this overexpression to selectively kill cancer cells. Herein, we investigate the efficacy of PAC-1 against uveal melanoma cell lines and report the synergistic combination of PAC-1 and entrectinib. This preclinical activity, tolerability data in mice, and the known clinical effectiveness of these drugs in human cancer patients led to a small Phase 1b study in patients with metastatic uveal melanoma. The combination of PAC-1 and entrectinib was tolerated with no treatment-related grade ≥3 toxicities in these patients. The pharmacokinetics of entrectinib were not affected by PAC-1 treatment. In this small and heavily pretreated initial cohort, stable disease was observed in four out of six patients, with a median progression-free survival of 3.38 months (95% CI 1.6-6.5 months). This study is an initial demonstration that the combination of PAC-1 and entrectinib may warrant further clinical investigation. Clinical trial registration: Clinical Trials.gov: NCT04589832.

Phase I dose-escalation study of procaspase-activating compound-1 in combination with temozolomide in patients with recurrent high-grade astrocytomas.

Background: Procaspase-3 (PC-3) is overexpressed in various tumor types, including gliomas. Targeted PC-3 activation combined with chemotherapy is a novel strategy for treating patients with high-grade gliomas, with promising preclinical activity. This study aimed to define safety and tolerability of procaspase-activating compound-1 (PAC-1) in combination with temozolomide (TMZ) for patients with recurrent high-grade astrocytomas. Methods: A modified-Fibonacci dose-escalation 3 + 3 design was used. PAC-1 was administered at increasing dose levels (DL; DL1 = 375 mg) on days 1-21, in combination with TMZ 150 mg/m2/5 days, per 28-day cycle. Dose-limiting toxicity was assessed during the first 2 cycles. Neurocognitive function (NCF) testing was conducted throughout the study. Results: Eighteen patients were enrolled (13 GBM, IDH-wild type; 2 astrocytoma, IDH-mutant, grade 3; 3 astrocytoma, IDH-mutant, grade 4). Dose escalation was discontinued after DL3 (ie, PAC-1, 625 mg) due to lack of additional funding. Grade 3 toxicity was observed in 1 patient at DL1 (elevated liver transaminases) and 1 at DL 2 (headache). Two partial responses were observed at DL1 in patients with GBM, O6-methylguanine-DNA methyltransferase (MGMT) promoter methylated. Two patients had stable disease, and 11 experienced progression. NCF testing did not show a clear relationship between PAC-1 dose, treatment duration, and declines in NCF. Conclusions: Combination of PAC-1 and TMZ was well tolerated up to 625 mg orally daily and TMZ orally 150 mg/m2/5 days per 28-day cycle. The maximum tolerated dose was not reached. Further dose escalation of PAC-1 in combination with TMZ is advised before conducting a formal prospective efficacy study in this patient population.

Single CAR-T cell treatment controls disseminated ovarian cancer in a syngeneic mouse model.

BACKGROUND: Treatment of some blood cancers with T cells that express a chimeric antigen receptor (CAR) against CD19 have shown remarkable results. In contrast, CAR-T cell efficacy against solid tumors has been difficult to achieve. METHODS: To examine the potential of CAR-T cell treatments against ovarian cancers, we used the mouse ovarian cancer cell line ID8 in an intraperitoneal model that exhibits disseminated solid tumors in female C57BL/6J mice. The CAR contained a single-chain Fv from antibody 237 which recognizes a Tn-glycopeptide-antigen expressed by ID8 due to aberrant O-linked glycosylation in the absence of the transferase-dependent chaperone Cosmc. The efficacy of four Tn-dependent CARs with varying affinity to Tn antigen, and each containing CD28/CD3ζ cytoplasmic domains, were compared in vitro and in vivo in this study. RESULTS: In line with many observations about the impact of aberrant O-linked glycosylation, the ID8Cosmc knock-out (ID8Cosmc-KO) exhibited more rapid tumor progression compared with wild-type ID8. Despite the enhanced tumor growth in vivo, 237 CAR and a mutant with 30-fold higher affinity, but not CARs with lower affinity, controlled advanced ID8Cosmc-KO tumors. Tumor regression could be achieved with a single intravenous dose of the CARs, but intraperitoneal administration was even more effective. The CAR-T cells persisted over a period of months, allowing CAR-treated mice to delay tumor growth in a re-challenge setting. The most effective CARs exhibited the highest affinity for antigen. Antitumor effects observed in vivo were associated with increased numbers of T cells and macrophages, and higher levels of cleaved caspase-3, in the tumor microenvironment. Notably, the least therapeutically effective CAR mediated tonic signaling leading to antigen-independent cytokine expression and it had higher levels of the immunosuppressive cytokine interleukin10. CONCLUSION: The findings support the development of affinity-optimized CAR-T cells as a potential treatment for established ovarian cancer, with the most effective CARs mediating a distinct pattern of inflammatory cytokine release in vitro. Importantly, the most potent Tn-dependent CAR-T cells showed no evidence of toxicity in tumor-bearing mice in a syngeneic, immunocompetent system.

Collagen-Anchored Interleukin-2 and Interleukin-12 Safely Reprogram the Tumor Microenvironment in Canine Soft-Tissue Sarcomas.

PURPOSE: Cytokine therapies such as IL2 and IL12 suffer from impractically small therapeutic windows driven by their on-target, off-tumor activity, limiting their clinical potential despite potent antitumor effects. We previously engineered cytokines that bind and anchor to tumor collagen following intratumoral injection, and sought to test their safety and biomarker activity in spontaneous canine soft-tissue sarcomas (STS). EXPERIMENTAL DESIGN: Collagen-binding cytokines were canine-ized to minimize immunogenicity and were used in a rapid dose-escalation study in healthy beagles to identify a maximum tolerated dose. Ten client-owned pet dogs with STS were then enrolled into trial, receiving cytokines at different intervals prior to surgical tumor excision. Tumor tissue was analyzed through IHC and NanoString RNA profiling for dynamic changes within treated tumors. Archived, untreated STS samples were analyzed in parallel as controls. RESULTS: Intratumorally administered collagen-binding IL2 and IL12 were well tolerated by STS-bearing dogs, with only Grade 1/2 adverse events observed (mild fever, thrombocytopenia, neutropenia). IHC revealed enhanced T-cell infiltrates, corroborated by an enhancement in gene expression associated with cytotoxic immune function. We found concordant increases in expression of counter-regulatory genes that we hypothesize would contribute to a transient antitumor effect, and confirmed in mouse models that combination therapy to inhibit this counter-regulation can improve responses to cytokine therapy. CONCLUSIONS: These results support the safety and activity of intratumorally delivered, collagen-anchoring cytokines for inflammatory polarization of the canine STS tumor microenvironment. We are further evaluating the efficacy of this approach in additional canine cancers, including oral malignant melanoma.

Canine as a Comparative and Translational Model for Human Mammary Tumor.

Despite the advances in research and treatment of human breast cancer, its incidence rate continues to increase by 0.5% per year, and the discovery of novel therapeutic strategies for specific subtypes of human breast cancer remains challenging. Traditional laboratory mouse models have contributed tremendously to human breast cancer research. However, mice do not develop tumors spontaneously; consequently, genetically engineered mouse models or patient-derived xenograft models are often relied upon for more sophisticated human breast cancer studies. Since human breast cancer develops spontaneously, there is a need for alternative, yet complementary, models that can better recapitulate the features of human breast cancer to better understand the molecular and clinical complexities of the disease in developing new therapeutic strategies. Canine mammary tumors are one such alternative model that share features with human breast cancer, including prevalence rate, subtype classification, treatment, and mutational profiles, all of which are described in this review.

Acute phase protein concentrations following serial procaine penicillin G injections in horses.

BACKGROUND: Acute phase protein (APP) measurement is used to detect inflammation. Intramuscular (IM) injections could cause tissue injury and induce an acute phase response (APR). OBJECTIVES: To evaluate the effects of IM procaine penicillin G (PPG) injections on APP concentrations in horses. STUDY DESIGN: Prospective longitudinal design. METHODS: PPG was administered intramuscularly to six horses, twice daily, for 5 days. Plasma fibrinogen (FIB), serum amyloid A (SAA), haptoglobin (HAP), creatine kinase (CK), and aspartate aminotransferase (AST) were quantified daily for 5 days before the first injection, during the course of administration, and for 4 days after the final dose. Analytes were quantified every other day for the remaining 16 days. Data were compared using a parametric or non-parametric repeated measures ANOVA and a Tukey's or Mann-Whitney rank sum test, respectively. Significance was set at p < 0.05. RESULTS: CK was increased over baseline (mean ± SD: 200 ± 74 IU/L) on Days 1-6 (p < 0.001 to p = 0.02, mean ± SD: 723-1177 ± 355-544 IU/L) and AST was increased above baseline (mean ± SD: 233 ± 58 IU/L) on Days 2-7 and 10 (p < 0.001 to p = 0.05, mean ± SD: 307-437 ± 79-146 IU/L). Increased FIB was noted over baseline (mean ± SD: 177 ± 30 mg/dl) on Days 6-8 and 10 (p = 0.02 to p = 0.03, mean ± SD: 234-252 ± 33-49 mg/dl). SAA was increased above baseline (mean ± SD: 4.7 ± 2.9) on Day 6 (p = 0.02, mean ± SD: 113 ± 186 µg/ml). There was no change in HAP. MAIN LIMITATIONS: Healthy horses were used, small sample size, and a lack of a negative control group. CONCLUSIONS: Serial intramuscular procaine penicillin G (IM PPG) injections may result in increased positive APP concentrations in horses and this must be considered when these test results are interpreted.

Phase I study of procaspase-activating compound-1 (PAC-1) in the treatment of advanced malignancies.

BACKGROUND: Procaspase-3 (PC-3) is overexpressed in multiple tumour types and procaspase-activating compound 1 (PAC-1) directly activates PC-3 and induces apoptosis in cancer cells. This report describes the first-in-human, phase I study of PAC-1 assessing maximum tolerated dose, safety, and pharmacokinetics. METHODS: Modified-Fibonacci dose-escalation 3 + 3 design was used. PAC-1 was administered orally at 7 dose levels (DL) on days 1-21 of a 28-day cycle. Dose-limiting toxicity (DLT) was assessed during the first two cycles of therapy, and pharmacokinetics analysis was conducted on days 1 and 21 of the first cycle. Neurologic and neurocognitive function (NNCF) tests were performed throughout the study. RESULTS: Forty-eight patients were enrolled with 33 completing ≥2 cycles of therapy and evaluable for DLT. DL 7 (750 mg/day) was established as the recommended phase 2 dose, with grade 1 and 2 neurological adverse events noted, while NNCF testing showed stable neurologic and cognitive evaluations. PAC-1's t1/2 was 28.5 h after multi-dosing, and systemic drug exposures achieved predicted therapeutic concentrations. PAC-1 clinical activity was observed in patients with neuroendocrine tumour (NET) with 2/5 patients achieving durable partial response. CONCLUSIONS: PAC-1 dose at 750 mg/day was recommended for phase 2 studies. Activity of PAC-1 in treatment-refractory NET warrants further investigation. CLINICAL TRIAL REGISTRATION: Clinical Trials.gov: NCT02355535.

Investigating PSMA differential expression in canine uroepithelial carcinomas to aid disease-based stratification and guide therapeutic selection.

BACKGROUND: In male dogs, uroepithelial cancers include invasive urothelial carcinoma (iUC) and prostate carcinoma (PCA). The inability to distinguish iUC involving the prostate from PCA results in indiscriminate clinical management strategies that could be suboptimal as first-line chemotherapy for iUC (cisplatin) and PCA (docetaxel) differ in people. Prostate specific membrane antigen (PSMA) is a transmembrane protein, and its overexpression has been identified in human prostate carcinoma and neovasculature associated with solid tumor growth. This study investigates whether differential PSMA expression exists between presumptive canine iUC and PCA among cell lines and archived patient samples, which might allow for improved accuracy in disease-based stratification and optimal chemotherapy selection. Additionally, in vitro sensitivities of reported canine iUC and PCA cell lines to uroepithelial directed chemotherapeutic agents were characterized. RESULTS: Normalized PSMA gene and protein expressions were not significantly different between 5 iUC and 4 PCA cell lines. PSMA protein expression was uniformly observed in uroepithelial cancers regardless of anatomic origin from archived patient samples, further confirming that PSMA cannot differentiate iUC from PCA. In vitro sensitivity of cell lines to uroepithelial directed chemotherapeutics revealed that vinblastine exerted the broadest cytotoxic activity. CONCLUSIONS: Differential expression of PSMA was not identified between canine iUC and PCA cell lines or archived patient samples, and PSMA alone cannot be used for disease stratification. Nonetheless given its conserved overexpression, PSMA may be a targetable surface marker for both canine iUC and PCA. Lastly, in uroepithelial carcinomas, vinblastine might exert the broadest anticancer activity regardless of cellular origin.

Comparative Oncology Assessment of a Novel Inhibitor of Valosin-Containing Protein in Tumor-Bearing Dogs.

Pet dogs with naturally occurring cancers play an important role in studies of cancer biology and drug development. We assessed tolerability, efficacy, and pharmacokinetic/pharmacodynamic relationships with a first-in-class small molecule inhibitor of valosin-containing protein (VCP/p97), CB-5339, administered to 24 tumor-bearing pet dogs. Tumor types assessed included solid malignancies, lymphomas, and multiple myeloma. Through a stepwise dose and schedule escalation schema, we determined the maximum tolerated dose to be 7.5 mg/kg when administered orally on a 4 days on, 3 days off schedule per week for 3 consecutive weeks. Adverse events were minimal and mainly related to the gastrointestinal system. Pharmacokinetic/pharmacodynamic data suggest a relationship between exposure and modulation of targets related to induction of the unfolded protein response, but not to tolerability of the agent. An efficacy signal was detected in 33% (2/6) of dogs with multiple myeloma, consistent with a mechanism of action relating to induction of proteotoxic stress in a tumor type with abundant protein production. Clinical trials of CB-5339 in humans with acute myelogenous leukemia and multiple myeloma are ongoing.

Target-Agnostic P-Glycoprotein Assessment Yields Strategies to Evade Efflux, Leading to a BRAF Inhibitor with Intracranial Efficacy.

The blood-brain barrier (BBB) presents a major hurdle in the development of central nervous system (CNS) active therapeutics, and expression of the P-glycoprotein (P-gp) efflux transporter at the blood-brain interface further impedes BBB penetrance of most small molecules. Designing efflux liabilities out of compounds can be laborious, and there is currently no generalizable approach to directly transform periphery-limited agents to ones active in the CNS. Here, we describe a target-agnostic, prospective assessment of P-gp efflux using diverse compounds. Our results demonstrate that reducing the molecular size or appending a carboxylic acid in many cases enables evasion of P-gp efflux in cell-based experiments and in mice. These strategies were then applied to transform a periphery-limited V600EBRAF inhibitor, dabrafenib, into versions that possess potent and selective anti-cancer activity but now also evade P-gp-mediated efflux. When compared to dabrafenib, the compound developed herein (everafenib) has superior BBB penetrance and superior efficacy in an intracranial mouse model of metastatic melanoma, suggesting it as a lead candidate for the treatment of melanoma metastases to the brain and gliomas with BRAF mutation. More generally, the results described herein suggest the actionability of the trends observed in these target-agnostic efflux studies and provide guidance for the conversion of non-BBB-penetrant drugs into versions that are BBB-penetrant and efficacious.

Three-dimensional hydrogel culture systems support growth and determination of chemosensitivity of feline sarcoma and carcinoma cell lines.

OBJECTIVE: To evaluate feline injection site-associated sarcoma (FISAS) and oral squamous cell carcinoma (FOSCC) cells in 3-D hydrogel-based cell cultures to determine chemosensitivity to carboplatin at concentrations comparable to those eluted from carboplatin-impregnated calcium sulfate hemihydrate (C-ICSH) beads. SAMPLE: 2 immortalized cell lines, each from a histologically confirmed primary FISAS and FOSCC. PROCEDURES: Hydrogels (10% wt/vol) were formed via UV exposure from methacrylamide-functionalized gelatin dissolved in PBSS. For each cell line, approximately 100,000 cells were encapsulated per hydrogel. Three cell-seeded 3-D hydrogels were evaluated for each carboplatin concentration (0, 150, 300, 450, and 600 µM) across 3 experiments. Drug efficacy was assessed by luminescence assay 72 hours after treatment. Growth of tumor cells treated with 300 µM or 600 µM carboplatin was evaluated using live-cell morphology imaging and confocal microscopy at 3, 7, and 14 days after treatment. RESULTS: Mean half-maximal inhibitory concentration (IC50) values for FISAS and FOSCC cells ranged from 123 to 171 µM and 155 to 190 µM, respectively, based on luminescence assay. Viability at 3, 7, and 14 days for both cell lines at 300 µM carboplatin was 50%, 25%, and 5% and at 600 µM carboplatin was 25%, 10%, and < 5%. CLINICAL RELEVANCE: 3-D hydrogel cell culture systems supported growth of feline tumor cells for determination of in vitro chemosensitivity. IC50s of each cell line were within the range of carboplatin concentrations eluted from C-ICSH beads. Cells from FISAS and FOSCC cell lines treated with carboplatin showed dose-dependent and time-dependent decreases in viability.

Three-dimensional hydrogel culture systems support growth and determination of chemosensitivity of feline sarcoma and carcinoma cell lines.

OBJECTIVE: To evaluate feline injection site-associated sarcoma (FISAS) and oral squamous cell carcinoma (FOSCC) cells in 3-D hydrogel-based cell cultures to determine chemosensitivity to carboplatin at concentrations comparable to those eluted from carboplatin-impregnated calcium sulfate hemihydrate (C-ICSH) beads. SAMPLE: 2 immortalized cell lines, each from a histologically confirmed primary FISAS and FOSCC. PROCEDURES: Hydrogels (10% wt/vol) were formed via UV exposure from methacrylamide-functionalized gelatin dissolved in PBSS. For each cell line, approximately 100,000 cells were encapsulated per hydrogel. Three cell-seeded 3-D hydrogels were evaluated for each carboplatin concentration (0, 150, 300, 450, and 600 µM) across 3 experiments. Drug efficacy was assessed by luminescence assay 72 hours after treatment. Growth of tumor cells treated with 300 µM or 600 µM carboplatin was evaluated using live-cell morphology imaging and confocal microscopy at 3, 7, and 14 days after treatment. RESULTS: Mean half-maximal inhibitory concentration (IC50) values for FISAS and FOSCC cells ranged from 123 to 171 µM and 155 to 190 µM, respectively, based on luminescence assay. Viability at 3, 7, and 14 days for both cell lines at 300 µM carboplatin was 50%, 25%, and 5% and at 600 µM carboplatin was 25%, 10%, and < 5%. CLINICAL RELEVANCE: 3-D hydrogel cell culture systems supported growth of feline tumor cells for determination of in vitro chemosensitivity. IC50s of each cell line were within the range of carboplatin concentrations eluted from C-ICSH beads. Cells from FISAS and FOSCC cell lines treated with carboplatin showed dose-dependent and time-dependent decreases in viability.

The use of alkaline phosphatase and runx2 to distinguish osteosarcoma from other common malignant primary bone tumors in dogs.

In dogs, primary bone tumors can be difficult to distinguish with histopathology. Of those tumors, osteosarcoma (OSA) is the most common and aggressive. In this study, 4 immunohistochemistry markers-alkaline phosphatase (ALP), osteonectin (ON), osteopontin (OP), and runx2-were evaluated for their ability to distinguish OSA from other primary bone tumors. The 42 formalin-fixed, paraffin-embedded, primary canine bone tumors included 15 OSAs, 8 chondrosarcomas, 11 fibrosarcomas, and 8 histiocytic sarcomas. All 4 antibodies were highly sensitive for detection of osteosarcoma. ALP was the most sensitive at 100% and runx2 the most specific at 78%. Running ALP and runx2 in series resulted in a sensitivity of 87% and a specificity of 85%. This combination of immunomarkers resulted in a diagnostic panel for distinguishing osteosarcoma from other primary bone tumors.