Breast cancer patient-derived explant cultures recapitulate in vivo drug responses

Assessment of drug sensitivity in tumor tissue ex vivo may significantly contribute to functional diagnostics to guide personalized treatment of cancer. Tumor organoid- and explant-cultures have become attractive tools towards this goal, although culturing conditions for breast cancer (BC) tissue have been among the most challenging to develop. Validation of possibilities to detect concordant responses in individual tumors and their respective cultures ex vivo is still needed. Here we employed BC patient-derived xenografts (PDXs) with distinct drug sensitivity, to evaluate different conditions for tissue dissociation, culturing and monitoring of treatment efficacy ex vivo, aiming to recapitulate the in vivo drug responses. The common challenge of discriminating between tumor and normal cells in the cultured tissue was also addressed. Following conventional enzymatic dissociation of BC tissue, the tumor cells stayed within the non-disrupted tissue fragments, while the single cells represented mostly normal host cells. By culturing such fragments as explants, viable tumor tissue could be maintained and treated ex vivo, providing representative indications on efficacy of the tested treatment. Thus, drug sensitivity profiles, including acquired chemoresistance seen in the PDXs, were recapitulated in the respective explants. To detect the concordant responses, however, the effect monitoring had to be harmonized with the characteristics of the cultured tissue. In conclusion, we present the feasibility of BC explants ex vivo to capture differences in drug sensitivity of individual tumors. The established protocols will aid in setting up an analogous platform for BC patient biopsies with the aim to facilitate functional precision medicine

Prognostic significance of S100A4-expression and subcellular localization in early-stage breast cancer

Purpose Prognostic factors are useful in order to identify early-stage breast cancer patients who might benefit from adjuvant treatment. The metastasis-promoting protein S100A4 has previously been associated with poor prognosis in breast cancer patients. The protein is expressed in diverse subcellular compartments, including the cytoplasm, extracellular space, and nucleus. Nuclear expression is an independent predictor of poor outcome in several cancer types, but the significance of subcellular expression has not yet been assessed in breast cancer. Methods Nuclear and cytoplasmic expression of S100A4 was assessed by immunohistochemistry in prospectively collected tumor samples from early-stage breast cancer patients using tissue microarrays. Results In patients not receiving adjuvant systemic therapy, nuclear or cytoplasmic expression was found in 44/291 tumors (15%). Expression of either nuclear or cytoplasmic S100A4 was associated with histological grade III, triple-negative subtype, and Ki-67-expression. Patients with S100A4-positive tumors had inferior metastasis-free and overall survival compared to S100A4-negative. When expression was analyzed separately, nuclear S100A4 was a significant predictor of outcome, while cytoplasmic was not. In patients who received adjuvant treatment 23/300 tumors (8%) were S100A4-positive, but no tumors displayed nuclear staining alone. S100A4-expression was strongly associated with histological grade III and triple-negative subtype. Although not significant, metastasis-free and overall survival was numerically reduced in patients with S100A4-positive tumors. Conclusion S100A4-expression was associated with poor outcome in early-stage breast cancer, but the low percentage of positive tumors and the modest survival differences imply that the clinical utility in selection of patients for adjuvant treatment is limited. This is a post-peer-review, pre-copyedit version of an article published in Breast Cancer Research and Treatment. The final authenticated version is available online at: http://dx.doi.org/10.1007/s10549-016-4096-

"Stroma-induced phenotypic plasticity offers phenotype-specific targeting to improve melanoma treatment".

Cancer cells' phenotypic plasticity, promoted by stromal cells, contributes to intra-tumoral heterogeneity and affects response to therapy. We have disclosed an association between fibroblast-stimulated phenotype switching and resistance to the clinically used BRAF inhibitor (BRAFi) vemurafenib in malignant melanoma, revealing a challenge in targeting the fibroblast-induced phenotype. Here we compared molecular features and drug sensitivity in melanoma cells grown as co-cultures with fibroblasts versus mono-cultures. In the presence of fibroblasts, melanoma cells switched to the dedifferentiated, mesenchymal-like, inflammatory phenotype that showed reduced sensitivity to the most of 275 tested cancer drugs. Fibroblasts, however, sensitized melanoma cells to PI3K inhibitors (PI3Ki) and particularly the inhibitor of GSK3, AR-A014418 (GSK3i), that showed superior efficacy in co-cultures. The proteome changes induced by the BRAFi+GSK3i combination mimicked changes induced by BRAFi in mono-cultures, and GSK3i in co-cultures. This suggests that the single drug drives the response to the combination treatment, depending on fibroblast presence or absence, consequently, phenotype. We propose that the BRAFi and GSK3i (or PI3Ki) combination exemplifies phenotype-specific combinatorial treatment that should be beneficial in phenotypically heterogeneous tumors rich in stromal interactions

Stroma-induced phenotypic plasticity offers phenotype-specific targeting to improve melanoma treatment

Cancer cells’ phenotypic plasticity, promoted by stromal cells, contributes to intra-tumoral heterogeneity and affects response to therapy. We have disclosed an association between fibroblast-stimulated phenotype switching and resistance to the clinically used BRAF inhibitor (BRAFi) vemurafenib in malignant melanoma, revealing a challenge in targeting the fibroblast-induced phenotype. Here we compared molecular features and drug sensitivity in melanoma cells grown as co-cultures with fibroblasts versus mono-cultures. In the presence of fibroblasts, melanoma cells switched to the dedifferentiated, mesenchymal-like, inflammatory phenotype that showed reduced sensitivity to the most of 275 tested cancer drugs. Fibroblasts, however, sensitized melanoma cells to PI3K inhibitors (PI3Ki) and particularly the inhibitor of GSK3, AR-A014418 (GSK3i), that showed superior efficacy in co-cultures. The proteome changes induced by the BRAFi+GSK3i combination mimicked changes induced by BRAFi in mono-cultures, and GSK3i in co-cultures. This suggests that the single drug drives the response to the combination treatment, depending on fibroblast presence or absence, consequently, phenotype. We propose that the BRAFi and GSK3i (or PI3Ki) combination exemplifies phenotype-specific combinatorial treatment that should be beneficial in phenotypically heterogeneous tumors rich in stromal interactions

Liver X receptors induce antiproliferative effects in basal‐like breast cancer

Liver X receptors (LXRs) are nuclear transcription factors important in the regulation of cholesterol transport, and glucose and fatty acid metabolism. The antiproliferative role of LXRs has been studied in a variety of malignancies and may represent a therapeutic opportunity in cancers lacking targeted therapies, such as triple‐negative breast cancer. In this study, we investigated the impact of LXR agonists alone and in combination with carboplatin in preclinical models of breast cancer. In vitro experiments revealed a dose‐dependent decrease in tumor cell proliferation in estrogen receptor‐positive breast cancer cells, whereas LXR activation in vivo resulted in an increased growth inhibitory effect in a basal‐like breast cancer model (in combination with carboplatin). Functional proteomic analysis identified differences in protein expression between responding and nonresponding models related to Akt activity, cell‐cycle progression, and DNA repair. Furthermore, pathway analysis suggested that the LXR agonist in combination with carboplatin inhibits the activity of targets of E2F transcription factors and affects cholesterol homeostasis in basal‐like breast cancer