Radiosensitization by the histone deacetylase inhibitor vorinostat under hypoxia and with capecitabine in experimental colorectal carcinoma

Background The histone deacetylase inhibitor vorinostat is a candidate radiosensitizer in locally advanced rectal cancer (LARC). Radiosensitivity is critically influenced by hypoxia; hence, it is important to evaluate the efficacy of potential radiosensitizers under variable tissue oxygenation. Since fluoropyrimidine-based chemoradiotherapy (CRT) is the only clinically validated regimen in LARC, efficacy in combination with this established regimen should be assessed in preclinical models before a candidate drug enters clinical trials. Methods Radiosensitization by vorinostat under hypoxia was studied in four colorectal carcinoma cell lines and in one colorectal carcinoma xenograft model by analysis of clonogenic survival and tumor growth delay, respectively. Radiosensitizing effects of vorinostat in combination with capecitabine were assessed by evaluation of tumor growth delay in two colorectal carcinoma xenografts models. Results Under hypoxia, radiosensitization by vorinostat was demonstrated in vitro in terms of decreased clonogenicity and in vivo as inhibition of tumor growth. Adding vorinostat to capecitabine-based CRT increased radiosensitivity of xenografts in terms of inhibited tumor growth. Conclusions Vorinostat sensitized colorectal carcinoma cells to radiation under hypoxia in vitro and in vivo and improved therapeutic efficacy in combination with capecitabine-based CRT in vivo. The results encourage implementation of vorinostat into CRT in LARC trials

Intraperitoneal Paclitaxel Is a Safe and Effective Therapeutic Strategy for Treating Mucinous Appendiceal Adenocarcinoma

Appendiceal adenocarcinomas (AAs) are a rare and heterogeneous mix of tumors for which few preclinical models exist. The rarity of AA has made performing prospective clinical trials difficult, which has partly contributed to AA remaining an orphan disease with no chemotherapeutic agents approved by the FDA for its treatment. AA has a unique biology in which it frequently forms diffuse peritoneal metastases but almost never spreads via a hematogenous route and rarely spreads to lymphatics. Given the localization of AA to the peritoneal space, intraperitoneal (IP) delivery of chemotherapy could be an effective treatment strategy. Here, we tested the efficacy of paclitaxel given by IP administration using three orthotopic patient-derived xenograft (PDX) models of AA established in immunodeficient NSG mice. Weekly IP paclitaxel treatment dramatically reduced AA tumor growth in all three PDX models. Comparing the safety and efficacy of intravenous (IV) to IP administration, IP delivery of paclitaxel was more effective with reduced systemic side effects in mice. Given the established safety record of IP paclitaxel in gastric and ovarian cancers, and lack of effective chemotherapeutics for AA, these data showing the activity of IP paclitaxel in orthotopic PDX models of mucinous AA support the evaluation of IP paclitaxel in a prospective clinical trial

Fibroblast-induced switching to the mesenchymal-like phenotype and PI3K/mTOR signaling protects melanoma cells from BRAF inhibitors

The knowledge on how tumor-associated stroma influences efficacy of anti-cancer therapy just started to emerge. Here we show that lung fibroblasts reduce melanoma sensitivity to the BRAF inhibitor (BRAFi) vemurafenib only if the two cell types are in close proximity. In the presence of fibroblasts, the adjacent melanoma cells acquire de-differentiated mesenchymal-like phenotype. Upon treatment with BRAFi, such melanoma cells maintain high levels of phospho ribosomal protein S6 (pS6), i.e. active mTOR signaling, which is suppressed in the BRAFi sensitive cells without stromal contacts. Inhibitors of PI3K/mTOR in combination with BRAFi eradicate pS6high cell subpopulations and potentiate anti-cancer effects in melanoma protected by the fibroblasts. mTOR and BRAF co-inhibition also delayed the development of early-stage lung metastases in vivo. In conclusion, we demonstrate that upon influence from fibroblasts, melanoma cells undergo a phenotype switch to the mesenchymal state, which can support PI3K/mTOR signaling. The lost sensitivity to BRAFi in such cells can be overcome by co-targeting PI3K/mTOR. This knowledge could be explored for designing BRAFi combination therapies aiming to eliminate both stroma-protected and non-protected counterparts of metastases

Peptide vaccine targeting mutated GNAS: A potential novel treatment for pseudomyxoma peritonei

Background Pseudomyxoma peritonei (PMP) is a rare, slow-growing abdominal cancer with no efficacious treatment options in non-resectable and recurrent cases. Otherwise, rare activating mutations in the GNAS oncogene are remarkably frequent in PMP and the mutated gene product, guanine nucleotide-binding protein α subunit (Gsα), is a potential tumor neoantigen, presenting an opportunity for targeting by a therapeutic cancer vaccine. Methods Tumor and blood samples were collected from 25 patients undergoing surgery for PMP (NCT02073500). GNAS mutation analysis was performed by next-generation targeted sequencing or digital droplet PCR. Responses to stimulation with Gsα mutated (point mutations R201H and R201C) 30 mer peptides were analyzed in peripheral blood T cells derived from patients with PMP and healthy donors. Fresh PMP tumor samples were analyzed by mass cytometry using a panel of 35 extracellular markers, and cellular subpopulations were clustered and visualized using the visual stochastic network embedding analysis tool. Results GNAS mutations were detected in 22/25 tumor samples (88%; R201H and R201C mutations detected in 16 and 6 cases, respectively). Strong T cell proliferation against Gsα mutated peptides was observed in 18/24 patients with PMP. Mass cytometry analysis of tumor revealed infiltration of CD3 +T cells in most samples, with variable CD4+:CD8 + ratios. A large proportion of T cells expressed immune checkpoint molecules, in particular programmed death receptor-1 and T cell immunoreceptor with Ig and ITIM, indicating that these T cells were antigen experienced. Conclusion These findings point to the existence of a pre-existing immunity in patients with PMP towards mutated Gsα, which has been insufficient to control tumor growth, possibly because of inhibition of antitumor T cells by upregulation of immune checkpoint molecules. The results form a rationale for exploring peptide vaccination with Gsα peptides in combination with immune checkpoint inhibiton as a possible curative treatment for PMP and other GNAS mutated cancers

Targeted Cancer Therapy with a Novel Anti-CD37 Beta-Particle Emitting Radioimmunoconjugate for Treatment of Non-Hodgkin Lymphoma.

177Lu-DOTA-HH1 (177Lu-HH1) is a novel anti-CD37 radioimmunoconjugate developed to treat non-Hodgkin lymphoma. Mice with subcutaneous Ramos xenografts were treated with different activities of 177Lu-HH1, 177Lu-DOTA-rituximab (177Lu-rituximab) and non-specific 177Lu-DOTA-IgG1 (177Lu-IgG1) and therapeutic effect and toxicity of the treatment were monitored. Significant tumor growth delay and increased survival of mice were observed in mice treated with 530 MBq/kg 177Lu-HH1 as compared with mice treated with similar activities of 177Lu-rituximab or non-specific 177Lu-IgG1, 0.9% NaCl or unlabeled HH1. All mice injected with 530 MBq/kg of 177Lu-HH1 tolerated the treatment well. In contrast, 6 out of 10 mice treated with 530 MBq/kg 177Lu-rituximab experienced severe radiation toxicity. The retention of 177Lu-rituximab in organs of the mononuclear phagocyte system was longer than for 177Lu-HH1, which explains the higher toxicity observed in mice treated with 177Lu-rituximab. In vitro internalization studies showed that 177Lu-HH1 internalizes faster and to a higher extent than 177Lu-rituximab which might be the reason for the better therapeutic effect of 177Lu-HH1

Internalization.

<p>Representative images of internalization of HH1 and Rituximab in Ramos cells after incubation with 10 μg/ml of HH1 or 20 μg/ml rituximab at 4°C or 37°C for 1 hour or 19 hours, respectively. HH1-DOTA bound to Alexa Fluor 488 is shown in green, Rituximab bound to Alexa Fluor 647 is shown in magenta and Hoechst 33342 bound to DNA in the cell nucleus is shown in blue. 20 to 40 cells were scanned for each treatment.</p

Hematology.

<p>Number of white blood cells (WBC), red blood cells (RBC) and platelets (PLT) in mice with Ramos xenografts treated with 530 MBq/kg ¹⁷⁷Lu-HH1, 530 MBq/kg ¹⁷⁷Lu-rituximab, 530 MBq/kg ¹⁷⁷Lu-IgG₁ non-specific isotype control, 15 μg/kg HH1, 0.9% NaCl (A) and 410 MBq/kg ¹⁷⁷Lu-HH1 and 300 MBq/kg ¹⁷⁷Lu-rituximab (B). The area of each circle represents the number of mice used to calculate the average values. Circles with dark rim and connected with lines represent samples taken at fixed time points during the study where all alive mice were sampled. Circles without dark rim and not connected by lines represent blood samples taken before euthanasia, where usually one or two mice were sacrificed. N = 1–10. Error bars = standard error. *: Significantly different from the corresponding 0.9% NaCl (control) group value (One-way ANOVA with Holm Sidak method for multiple comparisons, p < 0.05).</p

Overview of therapy and toxicity experiments in nude mice with Ramos xenografts.

<p>^aAt implantation of xenografts.</p><p>^bAt treatment injection Min-Max (Average ± SD).</p><p>Overview of therapy and toxicity experiments in nude mice with Ramos xenografts.</p

Incidence of main histological findings in nude mice with Ramos xenografts treated with 530 MBq/kg ¹⁷⁷Lu-HH1, ¹⁷⁷Lu-rituxmab and ¹⁷⁷Lu-IgG₁ non-specific isotype control or 0.9% NaCl.

<p>Incidence of main histological findings in nude mice with Ramos xenografts treated with 530 MBq/kg ¹⁷⁷Lu-HH1, ¹⁷⁷Lu-rituxmab and ¹⁷⁷Lu-IgG₁ non-specific isotype control or 0.9% NaCl.</p

Tumor growth, long term responders and survival of nude mice with Ramos xenografts after treatments with 410 MBq/kg ¹⁷⁷Lu-HH1, 300 MBq/kg ¹⁷⁷Lu-rituximab and 0.9% NaCl.

<p>(A) Individual tumor growth (n = Number of tumors in remission at the end of study/total number of initial tumors) (B) Percentage of long term responders; (C) Kaplan-Meier survival curve. End point: tumor volume ≥ 4 times initial tumor volume. Crosses represent censored animals that were euthanized before reaching 4 times the initial tumor volume. N = 5–6. Multiple comparisons were performed using Holm Sidak method. Significance level: p < 0.05.</p